Ultraviolet absorbing polymeric dyes and methods for using the same

ABSTRACT

Water soluble light harvesting multichromophores that have an ultraviolet absorption maximum are provided. In some embodiments, the multichromophores include a conjugated segment including a fused 6-5-6 tricyclic co-monomer and a UV absorbance-modifying co-monomer. The multichromophores may include an acceptor chromophore covalently linked to the multichromophore in energy-receiving proximity therewith. In some embodiments, a specific binding member is covalently linked to the multichromophore. Also provided are methods of evaluating a sample for the presence of a target analyte and methods of labelling a target molecule using compositions including the light harvesting multichromophores. Kits and systems for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/629,634, filed Jun. 21, 2017, granted U.S. Pat. No. 10,228,375, whichis a continuation of U.S. patent application Ser. No. 15/059,190, filedMar. 2, 2016, granted U.S. Pat. No. 9,719,998, which application,pursuant to 35 U.S.C. § 119 (e), claims priority 5 to the filing date ofU.S. Provisional Patent Application Ser. No. 62/132,449, filed Mar. 12,2015, the disclosures of which application are incorporated herein byreference.

INTRODUCTION

Fluorescent dyes are compounds which, when irradiated with light of awavelength which they absorb, emit light of a (usually) differentwavelength. Fluorescent dyes find use in a variety of applications inbiochemistry, biology and medicine, e.g. in diagnostic kits, inmicroscopy or in drug screening. Fluorescent dyes are characterized by anumber of parameters allowing a user to select a suitable dye dependingon the desired purpose. Parameters of interest include the excitationwavelength maximum, the emission wavelength maximum, the Stokes shift,the extinction coefficient, the fluorescence quantum yield and thefluorescence lifetime. Dyes may be selected according to the applicationof interest in order to, e.g., allow penetration of exciting radiationinto biological samples, to minimize background fluorescence and/or toachieve a high signal-to-noise ratio.

Molecular recognition involves the specific binding of two molecules.Molecules which have binding specificity for a target biomolecule finduse in a variety of research and diagnostic applications, such as thelabelling and separation of analytes, flow cytometry, in situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separations and chromatography. Targetbiomolecules may be detected by labelling with a fluorescent dye.

SUMMARY

Water soluble light harvesting multichromophores that have anultraviolet absorption maximum are provided. In some embodiments, themultichromophores include a conjugated segment including a fused 6-5-6tricyclic co-monomer and a UV absorbance-modifying co-monomer. Themultichromophores may include an acceptor chromophore covalently linkedto the multichromophore in energy-receiving proximity therewith. In someembodiments, a specific binding member is covalently linked to themultichromophore. Also provided are methods of evaluating a sample forthe presence of a target analyte and methods of labelling a targetmolecule using compositions including the light harvestingmultichromophores. Kits and systems for practicing the subject methodsare also provided.

BRIEF DESCRIPTION OF THE FIGURES

It is understood that the drawings, described below, are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 illustrates the fluorescence emission profiles of a variety ofpolymeric tandem dyes based on an exemplary multichromophore corestructure of the present disclosure linked to a variety of differentacceptor chromophores.

FIG. 2 illustrates the UV absorption spectra of a variety ofmultichromophores of interest, MC-1 to MC-5.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the term “sample” relates to a material or mixture ofmaterials, in some cases in liquid form, containing one or more analytesof interest. In some embodiments, the term as used in its broadestsense, refers to any plant, animal or bacterial material containingcells or producing cellular metabolites, such as, for example, tissue orfluid isolated from an individual (including without limitation plasma,serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents, as well as samples from theenvironment. The term “sample” may also refer to a “biological sample”.As used herein, the term “a biological sample” refers to a wholeorganism or a subset of its tissues, cells or component parts (e.g. bodyfluids, including, but not limited to, blood, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen). A “biological sample” canalso refer to a homogenate, lysate or extract prepared from a wholeorganism or a subset of its tissues, cells or component parts, or afraction or portion thereof, including but not limited to, plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors and organs. In certain embodiments, the sample hasbeen removed from an animal or plant. Biological samples may includecells. The term “cells” is used in its conventional sense to refer tothe basic structural unit of living organisms, both eukaryotic andprokaryotic, having at least a nucleus and a cell membrane. In certainembodiments, cells include prokaryotic cells, such as from bacteria. Inother embodiments, cells include eukaryotic cells, such as cellsobtained from biological samples from animals, plants or fungi.

As used herein, the terms “affinity” and “avidity” have the same meaningand may be used interchangeably herein. “Affinity” refers to thestrength of binding, increased binding affinity being correlated with alower Kd.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

As used herein, the terms “support bound” and “linked to a support” areused interchangeably and refer to a moiety (e.g., a specific bindingmember) that is linked covalently or non-covalently to a support ofinterest. Covalent linking may involve the chemical reaction of twocompatible functional groups (e.g., two chemoselective functionalgroups, an electrophile and a nucleophile, etc.) to form a covalent bondbetween the two moieties of interest (e.g. a support and a specificbinding member). In some cases, non-covalent linking may involvespecific binding between two moieties of interest (e.g., two affinitymoieties such as a hapten and an antibody or a biotin moiety and astreptavidin, etc.). In certain cases, non-covalent linking may involveabsorption to a substrate.

As used herein, the term “biomolecule” refers to an organic molecule ormacromolecule of a naturally occurring class of molecules, or aderivative thereof. Biomolecule is meant to encompass polypeptides(e.g., a peptide, an antibody or an antibody fragment), polynucleotides,carbohydrates (e.g., sugars) and lipids. In some cases, the biomoleculeis a specific binding member (e.g., as described herein).

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length,e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length.

As used herein, the term “isolated” refers to an moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 10 or more, such as 100 or more, 1000 or more, 10,000 or more,100,000 or more, 10⁶ or more, 10⁷ or more, 10⁸ or more or 10⁹ or moremembers.

Numeric ranges are inclusive of the numbers defining the range.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

As used herein, the term “specific binding” refers to the ability of acapture agent (or a first member of a specific binding pair) topreferentially bind to a particular analyte (or a second member of aspecific binding pair) that is present, e.g., in a homogeneous mixtureof different analytes. In some instances, a specific binding interactionwill discriminate between desirable and undesirable analytes in a samplewith a specificity of 10-fold or more for a desirable analyte over anundesirable analytes, such as 100-fold or more, or 1000-fold or more. Insome cases, the affinity between a capture agent and analyte when theyare specifically bound in a capture agent/analyte complex is at least10⁻⁸ M, at least 10⁻⁹M, such as up to 10⁻¹⁰ M.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

As used herein, the term “linker” or “linkage” refers to a linkingmoiety that connects two groups and has a backbone of 100 atoms or lessin length. A linker or linkage may be a covalent bond that connects twogroups or a chain of between 1 and 100 atoms in length, for example achain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbonatoms in length, where the linker may be linear, branched, cyclic or asingle atom. In some cases, the linker is a branching linker that refersto a linking moiety that connects three or more groups. In certaincases, one, two, three, four or five or more carbon atoms of a linkerbackbone may be optionally substituted with a sulfur, nitrogen or oxygenheteroatom. The bonds between backbone atoms may be saturated orunsaturated, and in some cases not more than one, two, or threeunsaturated bonds are present in a linker backbone. The linker mayinclude one or more substituent groups, for example with an alkyl, arylor alkenyl group. A linker may include, without limitations,polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, whichmay be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable.

As used herein, the terms “polyethylene oxide”, “PEO”, “polyethyleneglycol”, “PEG moiety” and “PEG” are used interchangeably and refer to apolymeric group including a chain described by the formula—(CH₂—CH₂—O—)_(n)— or a derivative thereof. In some embodiments, “n” is5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 orless, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, suchas 3 to 15, or 10 to 15. It is understood that the PEG polymeric groupmay be of any convenient length and may include a variety of terminalgroups and/or further substituent groups, including but not limited to,alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal and/orsubstituent groups. PEG groups that may be adapted for use in thesubject multichromophores include those PEGs described by S. Zalipsky in“Functionalized poly(ethylene glycol) for preparation of biologicallyrelevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165; andby Zhu et al in “Water-Soluble Conjugated Polymers for Imaging,Diagnosis, and Therapy”, Chem. Rev., 2012, 112 (8), pp 4687-4735.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Alkyl groups of interest include,but are not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group includes from 1 to 20 carbon atoms. In some embodiments, analkyl group includes from 1 to 10 carbon atoms. In certain embodiments,an alkyl group includes from 1 to 6 carbon atoms, such as from 1 to 4carbon atoms. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—),n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The terms “Alkynyl” and “alkyne” refer to straight or branchedmonovalent hydrocarbyl groups having from 2 to 6 carbon atoms andpreferably 2 to 3 carbon atoms and having at least 1 and preferably from1 to 2 sites of triple bond unsaturation. Examples of such alkynylgroups include acetylenyl (—C≡CH), and propargyl (—CH₂C≡CH). Unlessotherwise indicated, the terms include both substituted andunsubstituted groups.

The term “substituted alkynyl” “substituted alkynyl” refers to analkynyl group as defined herein having from 1 to 5 substituents, or from1 to 3 substituents, selected from alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,and —SO₂-heteroaryl.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system. Arylgroups of interest include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupincludes from 6 to 20 carbon atoms. In certain embodiments, an arylgroup includes from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Heteroarylgroups of interest include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene,triazole, xanthene, benzodioxole and the like. In certain embodiments,the heteroaryl group is from 5-20 membered heteroaryl. In certainembodiments, the heteroaryl group is from 5-10 membered heteroaryl. Incertain embodiments, heteroaryl groups are those derived from thiophene,pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline,imidazole, oxazole and pyrazine.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl andsubstituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Substituents of interest include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O⁻, —C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Forexample, a substituted group may bear a methylenedioxy substituent orone, two, or three substituents selected from a halogen atom, a(1-4C)alkyl group and a (1-4C)alkoxy group. When the group beingsubstituted is an aryl or heteroaryl group, the substituent(s) (e.g., asdescribed herein) may be referred to as “aryl substituent(s)”.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

As summarized above, water soluble light harvesting multichromophoresare provided. In some embodiments, the multichromophores include aconjugated segment including a fused 6-5-6 tricyclic co-monomer and a UVabsorbance-modifying co-monomer, where the multichromophore has anultraviolet absorption maximum. The multichromophores may include anacceptor chromophore covalently linked to the multichromophores inenergy-receiving proximity therewith. In some embodiments, a specificbinding member is covalently linked to the multichromophores. Alsoprovided are methods of evaluating a sample for the presence of a targetanalyte and methods of labelling a target molecule using compositionsincluding the light harvesting multichromophores. Kits and systems forpracticing the subject methods are also provided.

Before the various embodiments are described in greater detail, it is tobe understood that the teachings of this disclosure are not limited tothe particular embodiments described, and as such can, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present teachings will be limitedonly by the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

In further describing the subject invention, light harvestingmultichromophores are described first in greater detail. Next, tandemdyes and conjugates thereof which include the subject multichromophoresare described. Then, methods of interest in which compositions includingthe subject multichromophores find use are reviewed. Systems and kitsthat may be used in practicing methods of the invention are alsodescribed.

Light Harvesting Multichromophores

As summarized above, the present disclosure provides a light harvestingmultichromophore that has an ultraviolet absorption maximum. In someembodiments, the multichromophore includes a conjugated segment having:a fused 6-5-6 tricyclic co-monomer; and a UV absorbance-modifyingco-monomer; where the multichromophore has an ultraviolet absorptionmaximum. As used herein, the term “ultraviolet absorption maximum”refers to an absorption maximum wavelength that is in the UV region ofthe electromagnetic spectrum, e.g., a wavelength of 400 nm or less, suchas an absorption maximum wavelength in the range of 10 nm to 400 nm.

As used herein, the terms “light harvesting multichromophore”,“polymeric dye” and “conjugated polymer” are used interchangeably andrefer to a conjugated polymer which has a structure capable ofharvesting light with a particular absorption maximum wavelength andconverting it to emitted light at a longer emission maximum wavelength.In some cases, the light harvesting multichromophore is itselffluorescent. Conjugated polymers (CPs) are characterized by adelocalized electronic structure and may have an effective conjugationlength that is substantially shorter than the length of the polymerchain, because the backbone may contain a large number of conjugatedsegments in close proximity. In some cases, conjugated polymers areefficient for light harvesting and provide for optical amplification viaForster energy transfer to an acceptor.

As used herein the term “unit” refers to a structural subunit of apolymer. The term unit is meant to include monomers, co-monomers,co-blocks, conjugated segments, repeating units, and the like. A“repeating unit” is a subunit of a polymer that is defined by theminimum number of distinct structural features that are required for theunit to be considered monomeric, such that when the unit is repeated ntimes, the resulting structure describes the polymer or a block thereof.In some cases, the polymer may include two or more different repeatingunits, e.g., when the polymer is a multiblock polymer, each block maydefine a distinct repeating unit. In some cases, a repeating unit of thepolymer includes a single monomer group. In certain instances, arepeating unit of the polymer includes two or more monomer groups, i.e.,co-monomer groups, such as two, three, four or more co-monomer groups.As used herein, the term “co-monomer” or “co-monomer group” refers to astructural unit of a polymer that may itself be part of a repeating unitof the polymer. In some embodiments, the conjugated polymer includes ablock copolymer that is composed of blocks of polymerized monomers. Insuch cases, the block copolymer may be described as having distinctrepeating units each corresponding to a distinct co-block of thepolymer. In some cases, the polymer is a diblock copolymer that containstwo different co-blocks. In such cases, the polymer may be described asincluding co-blocks, where each co-block may be composed of co-monomers,such as one, two, three or more co-monomers.

As used herein, the term “UV absorbance-modifying co-monomer” refers toa co-monomer which imparts on the multichromophore an absorbance maximumthat is shifted to a shorter wavelength that is in the ultravioletregion (e.g., to a wavelength of 400 nm or less) relative to theabsorbance maximum of a control multichromophore, e.g., a conjugatedpolymer in which the UV absorbance-modifying co-monomer is not presentin a repeating unit. In some cases, the control multichromophore is apolyfluorene multichromophore. In some cases, the controlmultichromophore is a polycarbazole multichromophore.

Any convenient light harvesting multichromophores may be adapted toinclude an absorbance-modifying co-monomer in order to provide amultichromophore having an ultraviolet absorption maximum (e.g., anabsorption maximum at a wavelength of 400 nm or less). Light harvestingmultichromophores of interest that may be modified to include anabsorbance-modifying co-monomer include, but are not limited to, thosemultichromophores described by Gaylord et al. in US Publication Nos.20040142344, 20080293164, 20080064042, 20100136702, 20110256549,20120028828, 20120252986 and 20130190193 and U.S. Pat. Nos. 8,575,303and 8,802450, the disclosures of which Publications and Patents areherein incorporated by reference in their entirety; and Gaylord et al.,J. Am. Chem. Soc., 2001, 123 (26), pp 6417-6418; Feng et al., Chem. Soc.Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011,133 (32), pp 12600-12607, the disclosures of which are hereinincorporated by reference in their entirety.

In some embodiments, the multichromophores includes a plurality of firstoptically active units forming a conjugated system, having an absorptionwavelength (e.g., as described herein) at which the first opticallyactive units absorb light to form an excited state. In certaininstances, the multichromophores includes a conjugated polymer segmentor an oligomeric structure including bandgap-lowering n-conjugatedrepeating units.

The subject multichromophores may be water soluble. Any convenient watersolubilizing groups may be included in the multichromophore to providefor increased water-solubility. While the increase in solubility mayvary, in some instances the increase (as compared to the compoundwithout the WSG(s)) is 2 fold or more, e.g., 5 fold, 10 fold, 25 fold,50 fold, 100 fold or more. The term “water solubilizing group” (WSG)refers to a group that is well solvated in aqueous environments e.g.,under physiological conditions, and that imparts improved watersolubility upon the molecules to which it is attached. In someembodiments, a WSG increases the solubility of the multichromophore in apredominantly aqueous solution, as compared to a controlmultichromophore which lacks the WSG. The water solubilizing groups maybe any convenient hydrophilic group that is well solvated in aqueousenvironments. In some cases, the hydrophilic water solubilizing group ischarged, e.g., positively or negatively charged. In certain cases, thehydrophilic water solubilizing group is a neutral hydrophilic group. Insome embodiments, the WSG is a hydrophilic polymer, e.g., a polyethyleneglycol, a cellulose, a chitosan, or a derivative thereof. Watersolubilizing groups of interest include, but are not limited to,carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate,sulfonium, ester, polyethylene glycols (PEG) and modified PEGs,hydroxyl, amine, ammonium, guanidinium, pyridinium, polyamine andsulfonium, polyalcohols, straight chain or cyclic saccharides, primary,secondary, tertiary, or quaternary amines and polyamines, phosphonategroups, phosphinate groups, ascorbate groups, glycols, including,polyethers, —COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺, Y′, (CH₂CH₂O)_(p)R andmixtures thereof, where Y′ can be any halogen, sulfate, sulfonate, oroxygen containing anion, p can be 1 to 500, each R can be independentlyH or an alkyl (such as methyl) and M′ can be a cationic counterion orhydrogen, —(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy), —(CH₂CH₂O)_(yy)CH₂CH₂X—,—X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, and polyethylene glycol, wherein yy isselected from 1 to 1000, X is selected from O, S, and NR^(ZZ), andR^(ZZ) and R^(YY) are independently selected from H and C₁₋₃ alkyl.

Multiple WSGs may be included at a single location in the subjectmultichoromophores via a branching linker. In certain embodiments, thebranching linker is an aralkyl substitutent, further disubstituted withwater solubilizing groups. As such, in some cases, the branching linkergroup is a substitutent of the multichromophore that connects themultichromophore to two or more water solubilizing groups. In somecases, the incorporation of multiple WSGs via branching linkers impartsa desirable solubility on the multichromophore.

In some embodiments, the multichromophore includes substitutent(s)selected from, an alkyl, an aralkyl and a heterocyclic group, each groupfurther substituted with a include water solubilizing groups hydrophilicpolymer group, such as a polyethylglycol (PEG) (e.g., a PEG of 2-20units).

In some embodiments, the multichromophore is a water soluble lightharvesting multichromophore including a conjugated segment including: afluorene co-monomer;

and a UV absorbance-modifying co-monomer; wherein the multichromophorehas an ultraviolet absorption maximum. In some instances, themultichromophore is a water soluble light harvesting multichromophoreincluding a conjugated segment including: a carbazole co-monomer; and aUV absorbance-modifying co-monomer; wherein the multichromophore has anultraviolet absorption maximum. In certain embodiments, themultichromophore has an absorption maximum wavelength of 400 nm or less,such as a wavelength in the range of 10 nm to 400 nm, 100 nm to 400 nm,200 nm to 400 nm, 300 nm to 400 nm, 300 nm to 390 nm, 300 nm to 380 nm,300 nm to 370 nm, 300 nm to 360 nm, 300 nm to 350 nm, 300 nm to 340 nm,300 nm to 330 nm, or 300 nm to 325 nm. In certain embodiments, themultichromophore absorbs only UV light, i.e., only light of 400 nm orless, and does not absorb light at wavelengths of greater than 400 nm.In certain instances, the multichromophore has an absorption maximumwavelength of 300 nm to 400 nm. In some instances, the multichromophorehas an absorption maximum wavelength in the range of 300 to 400 nm (suchas in the range of 300 nm to 390 nm, 300 nm to 380 nm, 300 nm to 370 nm,300 nm to 360 nm or 300 nm to 325 nm) and an emission maximum wavelengthin the range of 375 to 900 nm (such as in the range of 380 nm to 900 nm,390 nm to 900 nm, or 400 nm to 900 nm). In certain cases, themultichromophore does not absorb visible light, e.g., no absorption atwavelengths of greater than 400 nm, such as wavelengths of 405 nm ormore. In certain instances, the multichromophore has an absorptionspectrum where 80% or more of the integrated absorption intensity (i.e.,the area under the absorption line), such as 85% or more, 90% or more,95% or more, 98% or more, or 99% or more, is in the UV region, atwavelengths of 400 nm or less.

The multichromophore may have any convenient length. In some cases, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 500,000, such as 2 to100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units orsegments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to100,000, or 500 to 50,000 units or segments. In some instances, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 1,000, such as 2 to500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to100, 9 to 100 or 10 to 100 units or segments.

The multichromophore may be of any convenient molecular weight (MW). Insome cases, the MW of the multichromophore may be expressed as anaverage molecular weight. the In some instances, the polymeric dye hasan average molecular weight of from 500 to 500,000, such as from 1,000to 100,000, from 2,000 to 100,000, from 10,000 to 100,000 or even anaverage molecular weight of from 50,000 to 100,000.

In some embodiments, the UV absorbance-modifying co-monomer constitutes25% or more by molarity of the multichromophore, such as 30% or more,40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or evenmore by molarity of the multichromophore. In such cases, themultichromophore may include 5 or more repeating units, such as 10 ormore, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 ormore, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more,1000 or more, 10,000 or more, or even more repeating units. In suchcases, the multichromophore may include 5 or more co-monomer units, suchas 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 ormore, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500or more, 1000 or more, 10,000 or more, or even more co-monomer units. Incertain embodiments, the UV absorbance-modifying co-monomer constitutes25% or more by molarity of the multichromophore, such as 30% or more,40% or more, 45% or more, 50% or more, or even more by molarity of themultichromophore, which includes 5 or more repeating units, such as 10or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70or more, 80 or more, 90 or more, 100 or more repeating units.

The subject multichromophore may have one or more desirablespectroscopic properties, such as a particular absorption maximumwavelength, a particular emission maximum wavelength, extinctioncoefficient, quantum yield, and the like. In some embodiments, themultichromophore has an emission maximum wavelength in the range of 300to 900 nm, such as 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to500 nm, 380 to 500 nm, 390 to 500 nm or 400 to 500 nm, where specificexamples of emission maxima of interest include, but are not limited to:395 nm±5 nm, 460 nm±5 nm, 490 nm±5 nm, 550 nm±5 nm, 560 nm±5 nm, 605nm±5 nm, 650 nm±5 nm, 680 nm±5 nm, 700 nm±5 nm, 805 nm±5 nm. In certaininstances, the multichromophore has an emission maximum wavelengthselected from 395 nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm,680 nm, 700 nm and 805 nm. In certain instances, the multichromophorehas an emission maximum wavelength of 395 nm±5 nm.

In some instances, the multichromophore has an extinction coefficient of5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹ or more, 7×10⁵ cm⁻¹M⁻¹ ormore, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ or more, such as 1×10⁶cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶ cm⁻¹M⁻¹ or more, 2.5×10⁶cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶ cm⁻¹M⁻¹ or more, 5×10⁶cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶ cm⁻¹M⁻¹ or more, or 8×10⁶cm⁻¹M⁻¹ or more. In such cases, the multichromophore may have 5 or morerepeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10or more, or even more repeating units. In some embodiments, themultichromophore has a molar extinction coefficient of 5×10⁵ M⁻¹cm⁻¹ ormore. In certain embodiments, the multichromophore has a molarextinction coefficient of 1×10⁶ M⁻¹cm⁻¹ or more.

In some instances, the multichromophore has an extinction coefficient of40,000 cm⁻¹M⁻¹ per repeating unit or more, such as 45,000 cm⁻¹M⁻¹ perrepeating unit or more, 50,000 cm⁻¹M⁻¹ per repeating unit or more,55,000 cm⁻¹M⁻¹ per repeating unit or more, 60,000 cm⁻¹M⁻¹ per repeatingunit or more, 70,000 cm⁻¹M⁻¹ per repeating unit or more, 80,000 cm⁻¹M⁻¹per repeating unit or more, 90,000 cm⁻¹M⁻¹ per repeating unit or more,100,000 cm⁻¹M⁻¹ per repeating unit or more, or even more. In someinstances, the 40,000 cm⁻¹M⁻¹ per repeating unit or more describedherein is an average extinction coefficient. In certain instances, therepeat unit of the multichromophore may include a single monomer, twoco-monomers, or three or more co-monomers. In some instances, themultichromophore has an extinction coefficient of 40,000 cm⁻¹M⁻¹ perco-monomer or more, such as 45,000 cm⁻¹M⁻¹ per co-monomer or more,50,000 cm⁻¹M⁻¹ per co-monomer or more, 55,000 cm⁻¹M⁻¹ per co-monomer ormore, 60,000 cm⁻¹M⁻¹ per co-monomer or more, 70,000 cm⁻¹M⁻¹ perco-monomer or more, 80,000 cm⁻¹M⁻¹ per co-monomer or more, 90,000cm⁻¹M⁻¹ per co-monomer or more, 100,000 cm⁻¹M⁻¹ per co-monomer or more,or even more. In some instances, the 40,000 cm⁻¹M⁻¹ per co-monomer ormore is an average extinction coefficient.

In certain cases, the multichromophore does not absorb visible light,e.g., light at greater than 400 nm, such as 405 nm or more. In certaininstances, the multichromophore has no significant absorption atwavelengths of greater than 400 nm, e.g., an extinction coefficient at awavelength of more than (such as 405 nm or more, e.g., 405 nm) of 1×10⁵cm⁻¹M⁻¹ or less, such as 9×10⁴ cm⁻¹M⁻¹ or less, 8×10⁴ cm⁻¹M⁻¹ or less,7×10⁴ cm⁻¹M⁻¹ or less, 6×10⁴ cm⁻¹M⁻¹ or less, 5×10⁴ cm⁻¹M⁻¹ or less,4×10⁴ cm⁻¹M⁻¹ or less, 3×10⁴ cm⁻¹M⁻¹ or less, 2×10⁴ cm⁻¹M⁻¹ or less,1×10⁴ cm⁻¹M⁻¹ or less, 5×10³ cm⁻¹M⁻¹ or less, 1×10³ cm⁻¹M⁻¹ or less, oreven less.

In certain embodiments, the multichromophore is a polymeric dye having aquantum yield of 0.3 or more, such as 0.35 or more, 0.4 or more, 0.45 ormore, 0.5 or more, 0.55 or more, 0.6 or more, 0.65 or more, 0.7 or more,or even more. In certain cases, the multichromophore has a quantum yieldof 0.4 or more. In certain instances, the polymeric dye has a quantumyield of 0.5 or more.

It is understood that in some cases the subject multichromophores mayinclude co-blocks (e.g., n and m co-blocks). The subjectmultichromophores may include any convenient linear arrangements of nand m co-blocks of various lengths within the structure of the overallpolymer. In addition, the multichromophores may include any convenientarrangements of co-monomers within such n and/or m co-blocks. A varietyof polymer synthesis methods may be utilized to prepare co-monomers andco-blocks of interest in the preparation of the subjectmultichromophores. It is understood that in some cases, thepolymerization methods may produce a composition including a populationof conjugated polymers that includes some variation with respect to theparticular length and/or terminal groups (i.e., end groups) present ineach CP of the population. The formulae depicted herein may refer to asingle compound or to a population or sub-population of polymericcompounds.

In some instances, the multichromophore is described by formula (I):

where:

F¹ is a fused 6-5-6 tricyclic co-monomer;

each M¹ and M² are each independently a UV absorbance-modifyingco-monomer;

b is 1 or 2;

a, c, d, e and f are each independently 0 or 1, wherein a+c+d+f≥1;

L¹ is a linking co-monomer comprising a chemoselective tag —Z¹;

each n is an integer from 1 to 100,000;

each m is 0 or an integer from 1 to 10,000;

p is an integer from 1 to 100,000; and

G¹ and G² are each independently selected from a terminal group, a πconjugated segment, a linker and a linked specific binding member.

In some instances of formula (I), F¹ is a fluorene co-monomer. In someinstances of formula (I), F¹ is a carbazole co-monomer. In some cases offormula (I), L¹ is a fluorene co-monomer. In certain embodiments offormula (I), L¹ is a carbazole co-monomer.

In some embodiments of formula (I), b is 1. In certain embodiments offormula (I), b is 2. In some instances of formula (I), a is 0. In somecases of formula (I), c is 0. In certain embodiments of formula (I), dis 0. In certain instances of formula (I), e is 0. In certain cases offormula (I), f is 0. In some embodiments of formula (I), a+c+d+f=1(i.e., a is 1, c is 1, d is 1 or f is 1). In some embodiments of formula(I), a+c+d+f=2. In some embodiments of formula (I), a+c+d+f=3. In someembodiments of formula (I), a+c+d+f=4. In some embodiments of formula(I), f is 1. In certain embodiments of formula (I), e is 1 and d or f is1, such that d+e+f=2. In certain instances of formula (I), e is 1 and dand f are each 0.

In certain embodiments of formula (I), e is 0 and d, f and m are each 0.In certain instances, e is 1, d+f≤1 and m≤1. In certain instances, e is1, d and f and each 0 and m≥1. In certain instances, e is 1; d+f=1 andm≥1. In some cases, d is 1 and f is 0. In some cases, d is 0 and f is 1.In some embodiments of formula (I), n, m and p are selected such thatthe multichromophore includes 2 to 100,000 repeat units (i.e., monomericrepeat units) in total, where the multichromophore may include a varietyof distinct monomeric repeat units. In some instances, when m is 0, p is1 and n is 2 to 100,000. In some embodiments of formula (I), L¹ is afluorene co-monomer.

A fused 6-5-6 tricyclic co-monomer is a co-monomer including a tricyclicaromatic group having three fused rings in the configuration 6-5-6, i.e.two benzo ring fused to a central 5 membered ring. The 5-membered ringmay be a carbocycle or a heterocycle and may further include a sidechainsubstituent at the ring atom that is not fused to a benzo ring. Incertain instances, the fused 6-5-6 tricyclic co-monomer is described bythe following structure:

where:

Z is —C(R¹)₂— or —N(R¹)—;

each R is independently H or one or more aryl substituents; and

each R¹ is independently selected from an alkyl, a substituted alkyl, anaralkyl, a substituted aralkyl, a PEG moiety and -L¹-Z¹, where L¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) or a WSG. As used in any of theformulae described herein, * denotes a site for covalent attachment tothe unsaturated backbone of a conjugated polymer or an end group. Insome embodiments, when Z is —N(R¹)—, the fused 6-5-6 tricyclicco-monomer is a carbazole co-monomer. Any convenient carbazoleco-monomers may be utilized in the subject multichromophores. In someembodiments, when Z is —C(R¹)₂—, the fused 6-5-6 tricyclic co-monomer isa fluorene co-monomer. Any convenient fluorene co-monomers may beutilized in the subject multichromophores. In certain instances of thefused 6-5-6 tricyclic co-monomer, each R¹ is selected from a benzylgroup substituted with one, two or more PEG moieties or an alkyl groupsubstituted with two or more PEG moieties.

A fluorene co-monomer is a co-monomer including an aromatic group havinga 9H-fluorene core structure substituted at the 9 position with anyconvenient sidechain substituent(s). In some cases, the fluoreneco-monomer is a 9,9-disubstituted fluorene. The fluorene co-monomer isconjugated to adjacent polymeric backbone groups via any convenientpositions of the fluorene core structure, such as any two positions ofpositions 1-8 (see numbering scheme below). In some embodiments, thefluorene core structure is linked to adjacent groups of the polymerbackbone via the 2 and 7 positions. In certain embodiments, the fluoreneco-monomer is described by the following structure:

where: each R¹ is independently selected from an alkyl, a substitutedalkyl, an aralkyl, a substituted aralkyl, a PEG moiety and -L¹-Z¹, whereL¹ is a linker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) or a WSG. In certain instances of thefluorene co-monomer, each R¹ is selected from a benzyl group substitutedwith one, two or more PEG moieties or an alkyl group substituted withtwo or more PEG moieties. The Z¹ functional group may find use incovalently linking the multichromophore to an acceptor chromophore(e.g., as described herein). In certain instances, Z¹ includes an aminogroup for covalently linking to the acceptor chromophore. In certaininstances, Z¹ includes an carboxylic acid group, or derivative thereof,for covalently linking to the acceptor chromophore. In certainembodiments, L¹ is a branched linker that links to two or more Z¹ groups(e.g., WSGs). In certain instances, the fluorene co-monomer is furthersubstituted with a R⁵ and/or R⁶ substituent located at one, two or morepositions selected from positions 1, 3, 4, 5, 6 and 8, where R⁵ and R⁶are independently selected from a water solubilizing group (WSG) and anaryl substituent (e.g., as described herein).

In certain instances, the fluorene co-monomer is described by thestructure:

where: each R¹ is as defined above; and R⁵ and R⁶ are independentlyselected from H, a water solubilizing group, or an aryl substituent(e.g., as described herein).

In some instances, the fluorene co-monomer is described by thestructure:

where each R² is a alkyl substituted with a water solubilizing group ora branched linker connected to two or more water solubilizing groups(e.g., a PEG-disubstituted benzyl or a PEG substituted alkyl). Incertain instances of the fluorene co-monomer, each R² is a benzyl groupsubstituted with one, two or three PEG moieties (e.g., —O(CH₂CH₂O)_(n)R′where R′ is H or an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16).In certain instances of the fluorene co-monomer, each R² is a benzylgroup substituted with one —O(CH₂CH₂O)_(n)R′ group (e.g., at the 2, 3 or4 position), where R′ is H or an alkyl and n is 1-20, e.g., 3-16 such asn is 8-16. In certain instances of the fluorene co-monomer, each R² is abenzyl group substituted with two —O(CH₂CH₂O)_(n)R′ groups (e.g., at the2,4-, 3,4- or 3,5-positions), where each R′ is independently H or analkyl and each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances of the fluorene co-monomer, each R² is a benzyl groupsubstituted with three —O(CH₂CH₂O)_(n)R′ groups (e.g., at the 2,4,6-,2,4,5- or 3,4,5-positions), where each R′ is independently H or an alkyland each n is independently 1-20, e.g., 3-16 such as n is 8-16. Incertain instances of the fluorene co-monomer, each R² is a lower alkylgroup substituted with a trivalent branching group each substituted withtwo PEG moieties (e.g., a −CO—NR″₂ or —O(CH₂R″)₂ trivalent branchinggroup where each R″ is independently a PEG moiety (e.g.,—O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 1-20, e.g., 3-16such as n is 8-16).

In certain embodiments, the fluorene co-monomer is described by thefollowing structure:

where R³ is an alkyl substituted with a water solubilizing group (e.g.,a PEG substituted alkyl), and R⁴ is L²-Z² wherein L² is a linker and Z²is a chemoselective tag (e.g., for conjugation to an acceptorchromophore). In some instances, the fluorene co-monomer is described bythe structure:

wherein:

R³ is a substituent comprising a water solubilizing group (e.g., asdescribed herein);

R⁴ is L²-Z² wherein L² is a linker and Z² is a chemoselective tag (e.g.,for conjugation to an acceptor chromophore); and

R⁵ and R⁶ are independently selected from H, a water solubilizing groupand an aryl substituent (e.g., an alkyl, a substituted alkyl, an alkoxy,a substituted alkoxy, a halogen or a nitro). In certain instances of thefluorene co-monomer, R³ is a lower alkyl group substituted with atrivalent branching group each substituted with two PEG moieties (e.g.,a —CO—NR″₂ or —O(CH₂R″)₂ trivalent branching group where each R″ is aPEG moiety (e.g., —O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is1-20, e.g., 3-16 such as n is 8-16).

Any of the fluorene co-monomers described above may be utilized in thesubject multichromophores, e.g., multichromophores of formulae (I)-(IV).In some cases, the multichromophores include, as part of the polymericbackbone, one of the following structures:

where each R³ is independently a water solubilizing group connected viaan optional linker, or an optionally substituted alkyl, aralkyl or arylgroup; Ar is an optionally substituted aryl or heteroaryl group; and nis an integer from 1 to 100,000. In certain embodiments, each R³ isindependently a substituted alkyl group. In certain embodiments, each R³is independently a substituted aralkyl group. In some cases, each R³ andeach Ar are independently substituted (via an optional linker) with awater solubilizing group, an acceptor chromophore, a chemoselectivefunctional group or a specific binding moiety.

As used herein, the terms “chemoselective functional group” and“chemoselective tag” are used interchangeably and refer to a functionalgroup that can selectively react with another compatible functionalgroup to form a covalent bond, in some cases, after optional activationof one of the functional groups. Chemoselective functional groups ofinterest include, but are not limited to, thiols and maleimide oriodoacetamide, amines and carboxylic acids or active esters thereof, aswell as groups that can react with one another via Click chemistry,e.g., azide and alkyne groups (e.g., cyclooctyne groups), as well ashydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne,phosphine, epoxide, and the like.

Any convenient linking co-monomers (L¹) may be incorporated into thesubject multichromophores to provide for a linking group to which may beattached any convenient moieties of interest. Linking co-monomers ofinterest include, but are not limited to, a fluorene co-monomer, aphenylenevinylene co-monomer, a phenyleneethynylene co-monomer, acarbazole co-monomer, a C₂-C₁₂ alkyne co-monomer, an arylene-ethynyleneco-monomer, a heteroarylene-ethynylene co-monomer, an arylene co-monomerand a heteroarylene co-monomer.

Any convenient chemoselective functional groups may be included in thesubject multichromophores (e.g., at the —Z¹), including, but are notlimited to, carboxylic acid, active ester (e.g., NHS or sulfo-NHSester), amino, hydroxyl, thiol, maleimide, iodoacetyl, hydrazido,hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, and thelike.

In certain cases, the linking co-monomer is a substituted orunsubstituted 1,4-phenyl, a substituted or unsubstituted 1,3-phenyl, asubstituted or unsubstituted 4,4′-biphenyl, a substituted orunsubstituted 2,5-pyridyl, and a substituted or unsubstituted2,6-pyridyl. In some instances, the linking co-monomer is a fluoreneco-monomer. In certain instances, the linking co-monomer is a UVabsorbance-modifying co-monomer (e.g., as described herein).

Any convenient end groups (e.g., G¹ and G²) may be utilized at theterminals of the subject multichromophores. G¹ and G² groups of interestinclude, but are not limited to a terminal capping group, a π conjugatedsegment, a linker and a linked specific binding member. In someembodiments, a terminal capping groups is a monovalent group which isconjugated to the backbone of the multichromophore after polymerization.In certain instances, the terminal capping group is an aryl, asubstituted aryl, a heteroaryl, a substituted heteroaryl, an alkyl or asubstituted alkyl. In certain cases, the terminal capping group isderived from a monomer used in the method of polymerization, e.g., aterminal group such as a halogen (e.g., Br), a boronic acid or a boronicester, which is capable of undergoing further conjugation. In someinstances, G¹ and/or G² is a t conjugated segment. As used herein, a tconjugated segment refers to any convenient segment of a conjugatedpolymer to which the multichromophore may be conjugated, i.e., allowingdelocalization of pi electron across adjacent units. In certainembodiments, G¹ and/or G² is a linker, such as a linker including afunctional group suitable for conjugation to a specific binding moiety.It is understood that linkers located at the G¹ and/or G² positions ofthe multichromophore may be selected so as to be orthogonal to any otherlinkers including chemoselective tags that may be present at a sidechainof the multichromophore (e.g., at Z¹). In certain embodiments, an aminofunctional group or derivative thereof is included at G¹ and/or G² and acarboxylic acid functional group or derivative thereof is included atZ¹. In certain embodiments, an carboxylic acid functional group orderivative thereof is included at G¹ and/or G² and an amino functionalgroup or derivative thereof is included at Z¹.

Any convenient UV absorbance-modifying co-monomers may be incorporatedinto the subject multichromophores to impart upon the multichromophore aUV absorption maximum. In some embodiments, the UV absorbance-modifyingco-monomers has an absorption maximum that is 350 nm or less, such as340 nm or less, 330 nm or less, 320 nm or less, 310 nm or less, 300 nmor less, 290 nm or less, 280 nm or less, 270 nm or less, 260 nm or less,250 nm or less, 240 nm or less, 230 nm or less, 220 nm or less, 210 nmor less, or 200 nm or less.

In some embodiments, the UV absorbance-modifying co-monomer is anoptionally substituted aryl or heteroaryl co-monomer. In some instances,the UV absorbance-modifying co-monomer is a substituted or unsubstitutedphenyl, biphenyl or pyridyl co-monomer. In certain embodiments, the UVabsorbance-modifying co-monomer is an optionally substituted aryl orheteroaryl co-monomer selected from substituted or unsubstituted1,4-phenyl, a substituted or unsubstituted 1,3-phenyl, a substituted orunsubstituted 4,4′-biphenyl, a substituted or unsubstituted 2,5-pyridyl,and a substituted or unsubstituted 2,6-pyridyl. In certain instances,the UV absorbance-modifying co-monomer is an optionally substituted arylor heteroaryl co-monomer selected from one of the following structures:

where Z²-Z⁵ are each independently CR or N, where at least one Z²-Z⁵ isN; and each R and each R¹¹-R¹⁶ are independently selected from hydrogen,water solubilizing group, halogen, cyano, alkoxy, substituted alkoxy,alkyl and substituted alkyl. In certain embodiments, one and only one ofZ²-Z⁵ is N. In certain embodiments, two and only two of Z²-Z⁵ is N. Incertain instances, R¹¹, R¹² and R¹⁴ are each H. In some instances, R¹²and R¹⁴ are each H. In some instances, R¹¹ and R¹³ are each H. In somecases, R¹⁵ and R¹⁶ are each H. In some instances, the halogen is fluoro.In certain instances, one and only one of R¹¹-R¹⁴ is an alkyl or asubstituted alkyl, and the other three of R¹¹-R¹⁴ are each H. In certaininstances, R¹¹ and R¹³ are each an alkyl or a substituted alkyl and R¹²and R¹⁴ are each H.

In some cases, the UV absorbance-modifying co-monomer is an optionallysubstituted aryl or heteroaryl co-monomer selected from one of thefollowing:

where n is 1-20 and R′ is H or lower alkyl. In some embodiments of thesubstituted aryl or heteroaryl co-monomer structures, n is an integerfrom 3 to 20.

In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

where n is 1-20 and R′ is H or lower alkyl. In certain instances, n is 3to 12.

In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

where each n is independently 1-20 and each R′ is independently H orlower alkyl. In certain embodiments of the substituted aryl orheteroaryl co-monomer structures shown above, n is 3. In certaininstances, R′ is methyl. In certain instances, R′ is hydrogen. In someembodiments, the multichromophore includes a substituted aryl co-monomerdescribed by the following structure:

wherein R¹¹ and R¹³ are each independently an alkyl or a substitutedalkyl, such as a lower alkyl or a substituted lower alkyl (e.g., asubstituted methyl, such as trifluoromethyl, difluoromethyl ormonofluoromethyl). In some embodiments, the multichromophore includes asubstituted aryl co-monomer described by the following structure:

wherein R¹¹ and R¹² are each independently an alkyl or a substitutedalkyl, such as a lower alkyl or a substituted lower alkyl (e.g., asubstituted methyl, such as trifluoromethyl, difluoromethyl ormonofluoromethyl). In some embodiments, the multichromophore includes asubstituted aryl co-monomer described by the following structure:

In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

wherein R¹¹ is an alkyl or a substituted alkyl, such as a lower alkyl ora substituted lower alkyl (e.g., a substituted methyl, such astrifluoromethyl, difluoromethyl or monofluoromethyl).

In some embodiments, the multichromophore includes a substituted arylco-monomer described by the following structure:

Any of the UV absorbance-modifying co-monomers described above may beutilized in the subject multichromophores, e.g., multichromophores offormulae (I)-(IV).

In some embodiments, the multichromophore is described by formula (II):

where F¹, M¹, M², a, b, c, e, L¹, Z¹, p, G¹ and G² are as described forformula (I). In some instances of formula (II), F¹ is a fluoreneco-monomer. In certain instances of formula (II), F¹ is a carbazoleco-monomer. In some embodiments of formula (II), L¹ is a fluoreneco-monomer. In certain embodiments of formula (II), L¹ is a carbazoleco-monomer. In certain embodiments, the multichromophore is described byformula (III):

where F¹, M¹, M², a, b, c, n, G¹ and G² are as described for formula(I). In some instances of formula (III), F¹ is a fluorene co-monomer. Incertain instances of formula (III), F¹ is a carbazole co-monomer.

In some instances of formulae (II) and (III), b is 1 and a+c≥1. Incertain instances of formulae (II) and (III), a+c=1 (e.g., a is 1 and cis 0, or a is 0 and c is 1). In certain embodiments of formulae (II) and(III), a+c=2. In some instances of formulae (II) and (III), G¹ is aterminal group; and G² is a terminal group, a linker or a linkedspecific binding member. In certain cases, G² is a linked specificbinding member. In some cases, G² is a linker, where the linker mayinclude a chemoselective tag.

In some instances, the multichromophore is described by formula (IV):

where F¹, M², b, c, e, f, L¹, Z¹, n, m, p, G¹ and G² are as describedfor formula (I). In some instances of formula (IV), F¹ is a fluoreneco-monomer. In certain instances of formula (IV), F¹ is a carbazoleco-monomer.

In some embodiments of formula (IV), b is 1; c is 0 or 1; e and f areeach 0 or 1, wherein e+f≥1; G¹ is a terminal group; and G² is a terminalgroup, a linker or a linked specific binding member. In certaininstances of formula (IV), c is 1. In certain cases of formula (IV), cis 0. In certain instances of formula (IV), e is 1. In certain cases offormula (IV), e is 0. In certain instances of formula (IV), f is 1. Incertain cases of formula (IV), f is 0. In certain cases, G² is a linkedspecific binding member. In some cases, G² is a linker, where the linkermay include a chemoselective tag. In some embodiments of formula (IV),L¹ is a fluorene co-monomer. In some embodiments of formula (IV), L¹ isa carbazole co-monomer.

In some embodiments, the multichromophore is described by the followingstructure:

where: each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n is an integer from 1 to 100,000. In some instances,G¹ is an aryl end group. In some cases, G¹ is a phenyl. In certainembodiments, Z is selected from a carboxylic acid, an amino or amaleimide. In certain instances, Z is a linked specific binding member.In some instances, L is an alkyl-carboxylic acid, such as —(CH₂)₃COOH.In certain embodiments, each R¹ group is a branching group furthersubstituted with two or more WSGs. In certain instances, the branchinggroup is a substituted aralkyl, such as a substituted benzyl group,which is substituted with two PEG groups.

In some embodiments, the multichromophore is described by the followingstructure:

where: each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n is an integer from 1 to 100,000. In some instances,G¹ is an aryl end group. In some cases, G¹ is a phenyl. In certainembodiments, Z is selected from a carboxylic acid, an amino or amaleimide. In certain instances, Z is a linked specific binding member.In some instances, L is an alkyl-carboxylic acid, such as —(CH₂)₃COOH.In certain embodiments, each R1 group is a branching group furthersubstituted with two or more WSGs. In certain instances, the branchinggroup is a substituted aralkyl, such as a substituted benzyl group,which is substituted with two PEG groups.

In some embodiments, the multichromophore is described by the followingstructure:

where: each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n is an integer from 1 to 100,000. In some instances,G¹ is an aryl end group. In some cases, G¹ is a phenyl. In certainembodiments, Z is selected from a carboxylic acid, an amino or amaleimide. In certain instances, Z is a linked specific binding member.In some instances, L is an alkyl-carboxylic acid, such as —(CH₂)₃COOH.In certain embodiments, each R1 group is a branching group furthersubstituted with two or more WSGs. In certain instances, the branchinggroup is a substituted aralkyl, such as a substituted benzyl group,which is substituted with two PEG groups.

In some embodiments, the multichromophore is described by the followingstructure:

where each Ak is independently an alkyl; mPEG is a methyl-capped PEGgroup where each n1 and n2 are independently 3 to 20; L is a linker andZ is a chemoselective tag or a linked specific binding member; G¹ is anend group; and n is an integer from 1 to 100,000. In some cases, G¹ is aphenyl. In certain embodiments, Z is selected from a carboxylic acid, anamino or a maleimide. In certain instances, Z is a linked specificbinding member. In some instances, L is an alkyl-carboxylic acid, suchas —(CH₂)₃COOH. In some embodiments, Ak is a C1-6 alkyl. In certaininstances, each n1 is 5 to 15. In certain instances, n2 is 3 to 12, suchas 3.

In some embodiments, the multichromophore is described by the followingstructure:

where mPEG is a methyl-capped PEG group; L is a linker and Z is achemoselective tag or a linked specific binding member; G¹ is an endgroup; and n is an integer from 1 to 100,000. In some cases, G¹ is aphenyl. In certain embodiments, Z is selected from a carboxylic acid, anamino or a maleimide. In certain instances, Z is a linked specificbinding member. In some instances, L is an alkyl-carboxylic acid, suchas —(CH₂)₃COOH.

In some embodiments, the multichromophore is described by the followingstructure:

where each Ak is independently an alkyl; mPEG is a methyl-capped PEGgroup where each n1 and each n2 are independently 3 to 20; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n is an integer from 1 to 100,000. In some cases, G¹is a phenyl. In certain embodiments, Z is selected from a carboxylicacid, an amino or a maleimide. In certain instances, Z is a linkedspecific binding member. In some instances, L is an alkyl-carboxylicacid, such as —(CH₂)₃COOH. In some embodiments, Ak is a C1-6 alkyl. Incertain instances, each n1 is 5 to 15. In certain instances, each n2 is3 to 12, such as 3.

In some embodiments, the multichromophore is described by the followingstructure:

where mPEG is a methyl-capped PEG group; L is a linker and Z is achemoselective tag or a linked specific binding member; G¹ is an endgroup; and n is an integer from 1 to 100,000. In some cases, G¹ is aphenyl. In certain embodiments, Z is selected from a carboxylic acid, anamino or a maleimide. In certain instances, Z is a linked specificbinding member. In some instances, L is an alkyl-carboxylic acid, suchas —(CH₂)₃COOH. In some cases, it is understood that in any of themultichromophore structures set forth herein, a fluorene co-monomer thatis depicted could be replaced with a corresponding carbazole co-monomerin the structure, such as a carbazole co-monomer including a singlesidechain group R¹ attached to the N atom of the carbazole co-monomer.Polymeric Tandem Dyes

In some embodiments, the light harvesting multichromophore is apolymeric tandem dye. Polymeric tandem dyes include two covalentlylinked moieties: a donor light harvesting multichromophore (e.g., asdescribed herein) and an acceptor chromophore. In certain embodiments,the polymeric tandem dye includes: a water soluble light harvestingmultichromophore including a conjugated segment including: a fused 6-5-6tricyclic co-monomer (e.g., as described herein); and a UVabsorbance-modifying co-monomer (e.g., as described herein); wherein themultichromophore has an ultraviolet absorption maximum (e.g., asdescribed herein); and an acceptor chromophore covalently linked to themultichromophore in energy-receiving proximity therewith. In someinstances, the acceptor chromophore is a quencher. In certain instances,the acceptor chromophore is a fluorescent dye. As used herein, the term“acceptor chromophore” refers to a light-absorbing molecule that iscapable of receiving or absorbing energy transferred from themultichromophore. In some cases, the acceptor chromophore can eitheremit as light the energy received from the multichromophore or dissipatethe energy as heat. It is understood that, unless otherwise stipulated,in the structures and formulae depicted herein, the label “dye” refersto an “acceptor chromophore”. As used herein, the term “quencher” refersto an acceptor chromophore that absorbs energy from the multichromophoreand does not emit light but rather can dissipate the energy as heat.

In some embodiments, the polymeric tandem dye may be excited in the UVregion at the absorption maximum wavelength of the donormultichromophore and may emit light at the emission wavelength of theacceptor chromophore. In some cases, the light-harvestingmultichromophore can transfer energy to an acceptor chromophore speciesin energy-receiving proximity. Mechanisms for energy transfer include,for example, resonant energy transfer (e.g., Forster (or fluorescence)resonance energy transfer, FRET), quantum charge exchange (Dexter energytransfer) and the like. In some instances, these energy transfermechanisms are relatively short range; that is, close proximity of thelight harvesting multichromophore system to the acceptor chromophoreprovides for efficient energy transfer. In some instances, underconditions for efficient energy transfer, amplification of the emissionfrom the acceptor chromophore occurs when the number of individualchromophores in the light harvesting multichromophore system is large;that is, the emission from the signaling chromophore is more intensewhen the incident light (the “pump light”) is at a wavelength which isabsorbed by the light harvesting multichromophore than when thesignaling chromophore is directly excited by the pump light.

By “efficient” energy transfer is meant 30% or more of the energyharvested is transferred to the acceptor. When the acceptor chromophoreis a fluorescent dye, the term efficient energy transfer refers to afluorescent quantum yield of 0.3 or more, such as 0.4 or more, 0.5 ormore, or even greater. By “amplification” is meant that the signal fromthe acceptor chromophore is 1.5× or greater when excited by the lightharvesting chromophore as compared to direct excitation with incidentlight of an equivalent intensity. The signal may be measured using anyconvenient method. In some cases, the 1.5× or greater signal refers toan intensity of emitted light. In certain cases, the 1.5× or greatersignal refers to an increased signal to noise ratio. In certainembodiments of the polymeric tandem dye, the acceptor chromophoreemission is 1.5 fold greater or more when excited by themultichromophore as compared to direct excitation of the acceptorchromophore with incident light.

In some instances, the polymeric tandem dye has an extinctioncoefficient of 5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹ or more,7×10⁵ cm⁻¹M⁻¹ or more, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ or more,such as 1×10⁶ cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶ cm⁻¹ M⁻¹or more, 2.5×10⁶ cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶ cm⁻¹M⁻¹or more, 5×10⁶ cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶ cm⁻¹M⁻¹ ormore, or 8×10⁶ cm⁻¹M⁻¹ or more. In some embodiments, the polymerictandem dye has a molar extinction coefficient of 5×10⁵ M⁻¹cm⁻¹ or more.In certain embodiments, the polymeric tandem dye has a molar extinctioncoefficient of 1×10⁶ M⁻¹cm⁻¹ or more.

In certain embodiments, the polymeric tandem dye has a quantum yield of0.3 or more, such as 0.35 or more, 0.4 or more, 0.45 or more, 0.5 ormore, 0.55 or more, 0.6 or more, 0.65 or more, 0.7 or more, or evenmore. In certain cases, the polymeric tandem dye has a quantum yield of0.4 or more. In certain instances, the polymeric tandem dye has aquantum yield of 0.5 or more.

Any convenient fluorescent dyes may be utilized in the polymeric tandemdyes as an acceptor chromophore. The terms “fluorescent dye” and“fluorophore” are used interchangeably herein. In some embodiments, theacceptor chromophore is a cyanine dye, a xanthene dye, a coumarin dye, athiazine dye or an acridine dye. Fluorescent dyes of interest include,but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red,tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700,7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665, BODIPY R6G, BODIPY TMR, BODIPYTR, conjugates thereof, and combinations thereof. Lanthanide chelates ofinterest include, but are not limited to, europium chelates, terbiumchelates and samarium chelates. In some embodiments, the polymerictandem dye includes a polymeric dye linked to an acceptor fluorophoreselected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 andAlexa700. In certain embodiments, the polymeric tandem dye includes apolymeric dye linked to an acceptor fluorophore selected from Dyomicsdyes (such as DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), BiotiumCF 555, Cy 3.5, and diethylamino coumarin.

In some embodiments, the polymeric tandem dye has an absorption maximumwavelength in the range of 300 to 400 nm and an emission maximumwavelength in the range of 375 to 900 nm. In certain embodiments, thepolymeric tandem dye includes a water soluble light harvestingmultichromophore having a conjugated segment including: a fused 6-5-6tricyclic co-monomer and a UV absorbance-modifying co-monomer. In someinstances, the UV absorbance-modifying co-monomer is present at 25% ormore by molarity in the polymeric tandem dye, where the multichromophoreis a conjugated polymer including 5 or more monomeric repeat units.

In some instances, the polymeric tandem dye is described by formula (V):

where:

F¹ is a fused 6-5-6 tricyclic co-monomer (e.g., as described herein);

each M¹ and M² are each independently a UV absorbance-modifyingco-monomer (e.g., as described herein);

b is 1 or 2;

a, c, d and f are each independently 0 or 1, wherein a+c+d+f≥1;

e is 1 or 2;

L¹ is a linking co-monomer that is linked to the acceptor chromophore—C¹;

n is an integer from 1 to 10,000;

m is an integer from 1 to 10,000;

p is an integer from 1 to 100,000; and

G¹ and G² are each independently selected from a terminal group, a πconjugated segment, a linker and a linked specific binding member. Incertain embodiments of formula (V), e is 1. In some instances of formula(V), d+f≤1 (i.e., d is 1 and f is 0 or d is 0 and f is 1) and m 1. Insome instances of formula (V), F¹ is a fluorene co-monomer. In certaininstances of formula (V), F¹ is a carbazole co-monomer.

In some embodiments, the polymeric tandem dye is described by formula(VI):

where F¹, M¹, M², a, b, c, e, L¹, C¹, p, G¹ and G² are as described forformula (V). In some instances of formula (VI), F¹ is a fluoreneco-monomer. In certain instances of formula (VI), F¹ is a carbazoleco-monomer. In some embodiments, the polymeric tandem dye is describedby formula (VII):

where F¹, M², b, c, e, f, n, m, L¹, C¹, p, G¹ and G² are as describedfor formula (V). In some instances of formula (VII), F¹ is a fluoreneco-monomer. In certain instances of formula (VII), F¹ is a carbazoleco-monomer. In certain instances of formula (VII), b is 1; c is 0 or 1;e is 1; and f is 0 or 1. In some cases of formula (VIII), c is 1. Insome embodiments of formula (VIII), c is 0. In some instances of formula(VIII), f is 1. In certain instances of formula (VIII), f is 0. Incertain cases of formula (VII), G¹ is a terminal group and G² is aterminal group, a linker or a linked specific binding member. In someinstances, G² is a linker that includes a chemoselective tag. In somecases, G² is a linked specific binding member.

Any convenient fluorene co-monomers (e.g., as described herein) may beutilized in the polymeric tandem dyes of formulae (V) to (VII). Anyconvenient carbazole co-monomers may be utilized in the polymeric tandemdyes of formulae (V) to (VII). Any convenient UV absorbance-modifyingco-monomers (e.g., as described herein) may be utilized in the polymerictandem dyes of formulae (V) to (VII). Any convenient linking co-monomers(e.g., as described herein) may be utilized in the polymeric tandem dyesof formulae (V) to (VII). In certain embodiments of formulae (V) to(VII), L¹ is a fluorene co-monomer. In certain embodiments of formulae(V) to (VII), L¹ is a carbazole co-monomer. In some instances offormulae (V) to (VII), L¹ is described by the structure:

wherein:

R¹ is a substituent including a water solubilizing group (e.g., a PEGsubstituted alkyl);

R² is L²-Z² wherein L² is a linker and Z² is the acceptor chromophore;and

R³ and R⁴ are independently selected from H, a water solubilizing group,an alkyl, a substituted alkyl, an alkoxy, a substituted alkoxy, ahalogen and a nitro. In certain instances, R³ and R⁴ are each hydrogen.

In some embodiments of formula (V) to (VIII), at least one of G¹ and G²is -L³-Z where L³ is a linker (e.g., as described herein) and Z is aspecific binding member (e.g., as described herein). In some embodimentsof formula (I) to (VIII), at least one of G¹ and G² is -L³-Z where L³ isa linker (e.g., as described herein) and Z is a chemoselective tag(e.g., as described herein). In some instances, Z is selected fromcarboxylic acid, active ester (e.g., N-hydroxy succinimidyl ester (NHS)or sulfo-NHS), amino, maleimide, iodoacetyl and thiol. In certainembodiments of formula (I) to (VIII), at least one of G¹ and G² isdescribed by the following structure:*—Ar-L-Z

where Ar is a π-conjugated aryl group, L is a linker and Z is achemoselective tag or a specific binding member. In certain embodimentsof formula (I) to (VIII), at least one of G¹ and G² is described by oneof the following structures:

wherein:

q is 0 or an integer from 1-12;

L is an optional linker; and

Z is a chemoselective tag or a specific binding member. In certainembodiments of formula (I) to (VIII), at least one L² group is describedby the structure:

where q is 0 or an integer from 1-12; L is an optional linker; and Z isa chemoselective tag or a specific binding member. In certain instances,—NH-L-Z includes an amide linkage to the chemoselective tag or specificbinding member. In certain embodiments, Z is a biomolecule. In certaininstances, Z is an antibody. In some instances, Z is an antibodyfragment or binding derivative thereof. In some cases, the antibodyfragment or binding derivative thereof is selected from a Fab fragment,a F(ab′)₂ fragment, a scFv, a diabody and a triabody.

In some embodiments of formulae (V) to (VII), the C¹ is selected from acyanine dye, a xanthene dye, a coumarin dye, a thiazine dye and anacridine dye, linked to L¹ via an optional linker. In certain cases, thelinker is selected from an alkyl, a substituted alkyl, an alkyl-amido,an alkyl-amido-alkyl and a PEG moiety. In certain embodiments offormulae (V) to (VII), the acceptor chromophore C¹ is selected from DY431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701,DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800,DY 831, Biotium CF 555, Cy 3.5 and diethylamino coumarin.

In some embodiments, the polymeric tandem dye is described by thefollowing structure:

where: each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs; R² is L²-Z²wherein L² is a linker and Z² is the acceptor chromophore; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n, m and p are each independently an integer from 1 to100,000. In some instances, G¹ is an aryl end group. In some cases, G¹is a phenyl. In certain embodiments, Z is selected from a carboxylicacid, an amino or a maleimide. In certain instances, Z is a linkedspecific binding member. In some instances, L is an alkyl-carboxylicacid, such as —(CH₂)₃COOH. In certain embodiments, one or more of the R¹groups is a branching group further substituted with two or more WSGs.In certain instances, the branching group is a substituted aralkyl, suchas a substituted benzyl group, which is disubstituted with two PEGgroups.

In some embodiments, the multichromophore is described by the followingstructure:

where: each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs; R² is L²-Z²wherein L² is a linker and Z² is the acceptor chromophore; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n, m and p are each independently an integer from 1 to100,000. In some instances, G¹ is an aryl end group. In some cases, G¹is a phenyl. In certain embodiments, Z is selected from a carboxylicacid, an amino or a maleimide. In certain instances, Z is a linkedspecific binding member. In some instances, L is an alkyl-carboxylicacid, such as —(CH₂)₃COOH. In certain embodiments, one or more of the R¹groups is a branching group further substituted with two or more WSGs.In certain instances, the branching group is a substituted aralkyl, suchas a substituted benzyl group, which is disubstituted with two PEGgroups.

In some embodiments, the multichromophore is described by the followingstructure:

where: each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs; R² is L²-Z²wherein L² is a linker and Z² is the acceptor chromophore; L is a linkerand Z is a chemoselective tag or a linked specific binding member; G¹ isan end group; and n is an integer from 1 to 100,000. In some instances,G¹ is an aryl end group. In some cases, G¹ is a phenyl. In certainembodiments, Z is selected from a carboxylic acid, an amino or amaleimide. In certain instances, Z is a linked specific binding member.In some instances, L is an alkyl-carboxylic acid, such as —(CH₂)₃COOH.In certain embodiments, one or more of the R¹ groups is a branchinggroup further substituted with two or more WSGs. In certain instances,the branching group is a substituted aralkyl, such as a substitutedbenzyl group, which is substituted with two PEG groups.

In some embodiments, the multichromophore is described by the followingstructure:

where each R¹ is independently selected from an alkyl substituted with aWSG, a branching group further substituted with two or more WSGs, and-Ak-O-mPEG_(n1); each Ak is independently an alkyl; mPEG is amethyl-capped PEG group where each n1 and n2 are independently 3 to 20;R² is L²-Z² wherein L² is a linker and Z² is the acceptor chromophore Lis a linker and Z is a chemoselective tag or a linked specific bindingmember; G¹ is an end group; and n is an integer from 1 to 100,000. Insome cases, G¹ is a phenyl. In certain embodiments, Z is selected from acarboxylic acid, an amino or a maleimide. In certain instances, Z is alinked specific binding member. In some instances, L is analkyl-carboxylic acid, such as —(CH₂)₃COOH. In some embodiments, Ak is aC₁₋₆ alkyl. In some instances, each Ak is —CH₂—. In some cases, each R¹is -propyl-O-mPEG₁₁. In certain instances, each n1 is 5 to 15. Incertain instances, n2 is 3 to 12, such as 3.

In some embodiments, the multichromophore is described by the followingstructure:

where each Ak is independently an alkyl; mPEG is a methyl-capped PEGgroup where each n1 and each n2 are independently 3 to 20; wherein L² isa linker and Z² is the acceptor chromophore; L is a linker and Z is achemoselective tag or a linked specific binding member; G¹ is an endgroup; and n is an integer from 1 to 100,000. In some cases, G¹ is aphenyl. In certain embodiments, Z is selected from a carboxylic acid, anamino or a maleimide. In certain instances, Z is a linked specificbinding member. In some instances, L is an alkyl-carboxylic acid, suchas —(CH₂)₃COOH. In some embodiments, Ak is a C1-6 alkyl. In certaininstances, each n1 is 5 to 15. In certain instances, each n2 is 3 to 12,such as 3. In some instances, the Ak of the UV-modifying co-monomer is—CH₂—. In some instances, the each Ak of the fluorene co-monomer is—(CH₂)₃—.

It is understood that for any of the structures and formula depictedherein that in some cases of the subject multichromophore the end groupsdepicted may be located at the opposite ends to those shown, e.g., theend groups G¹ and -Ph-L-Z may be switched.

Labelled Specific Binding Members

Aspects of the present disclosure include labelled specific bindingmembers. A labelled specific binding member is a conjugate of a subjectmultichromophore (e.g., as described herein) and a specific bindingmember. The multichromophore may be a polymeric dye. Themultichromophore may be polymeric tandem dye. Any of themultichromophores described herein may be conjugated to a specificbinding member. The specific binding member and the multichromophore maybe conjugated (e.g., covalently linked) to each other via any convenientlocations of the multichromophore, via an optional linker.

As used herein, the term “specific binding member” refers to one memberof a pair of molecules which have binding specificity for one another.One member of the pair of molecules may have an area on its surface, ora cavity, which specifically binds to an area on the surface of, or acavity in, the other member of the pair of molecules. Thus the membersof the pair have the property of binding specifically to each other toproduce a binding complex. In some embodiments, the affinity betweenspecific binding members in a binding complex is characterized by aK_(d) (dissociation constant) of 10⁻⁶ M or less, such as 10⁻⁷ M or less,including 10⁻⁸ M or less, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, including10⁻¹⁵ M or less. In some embodiments, the specific binding membersspecifically bind with high avidity. By high avidity is meant that thebinding member specifically binds with an apparent affinitycharacterized by an apparent K_(d) of 10×10⁻⁹ M or less, such as 1×10⁻⁹M or less, 3×10⁻¹⁰ M or less, 1×10⁻¹⁰ M or less, 3×10⁻¹¹ M or less,1×10⁻¹¹ M or less, 3×10⁻¹² M or less or 1×10⁻¹² M or less.

As used herein, the term “proteinaceous” refers to a moiety (e.g., aspecific binding member) that is composed of amino acid residues. Aproteinaceous moiety may be a polypeptide. In some embodiments, thespecific binding member is proteinaceous. In certain cases, theproteinaceous specific binding member is an antibody. In certainembodiments, the proteinaceous specific binding member is an antibodyfragment, e.g., a binding fragment of an antibody that specific binds toa polymeric dye. As used herein, the terms “antibody” and “antibodymolecule” are used interchangeably and refer to a protein consisting ofone or more polypeptides substantially encoded by all or part of therecognized immunoglobulin genes. The recognized immunoglobulin genes,for example in humans, include the kappa (k), lambda (I), and heavychain genetic loci, which together comprise the myriad variable regiongenes, and the constant region genes mu (u), delta (d), gamma (g), sigma(e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. An immunoglobulin light or heavy chain variable regionconsists of a “framework” region (FR) interrupted by three hypervariableregions, also called “complementarity determining regions” or “CDRs”.The extent of the framework region and CDRs have been precisely defined(see, “Sequences of Proteins of Immunological Interest,” E. Kabat etal., U.S. Department of Health and Human Services, (1991)). Thenumbering of all antibody amino acid sequences discussed herein conformsto the Kabat system. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs. The CDRs are primarily responsible for binding to an epitopeof an antigen.

The term antibody is meant to include full length antibodies and mayrefer to a natural antibody from any organism, an engineered antibody,or an antibody generated recombinantly for experimental, therapeutic, orother purposes as further defined below. Antibody fragments of interestinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, scFv, or otherantigen-binding subsequences of antibodies, either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies. Antibodies may be monoclonal or polyclonaland may have other specific activities on cells (e.g., antagonists,agonists, neutralizing, inhibitory, or stimulatory antibodies). It isunderstood that the antibodies may have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other antibody functions.

In certain embodiments, the specific binding member is an antibody. Incertain embodiments, the specific binding member is a Fab fragment, aF(ab′)₂ fragment, a scFv, a diabody or a triabody. In some cases, thespecific binding member is a murine antibody or binding fragmentthereof. In certain instances, the specific binding member is arecombinant antibody or binding fragment thereof.

In some embodiments, the labelled specific binding member includes: awater soluble light harvesting multichromophore comprising a conjugatedsegment including: a fluorene co-monomer (e.g., as described herein);and a UV absorbance-modifying co-monomer (e.g., as described herein);where the multichromophore has an ultraviolet absorption maximum; and aspecific binding member covalently linked to the multichromophore. Incertain embodiments of the labelled specific binding member, themultichromophore has an absorption maximum wavelength in the range of300 to 400 nm and an emission maximum wavelength in the range of 375 to900 nm. In some instances of the labelled specific binding member, theUV absorbance-modifying co-monomer includes 25% or more by molarity ofthe multichromophore; and the multichromophore is a conjugated polymerincluding 5 or more repeat units.

In certain instances of the labelled specific binding member, themultichromophore has a molar extinction coefficient of 5×10⁵ M⁻¹cm⁻¹ ormore (e.g., as described herein). In certain cases of the labelledspecific binding member, the multichromophore has a quantum yield of 0.3or more (e.g., as described herein). In some embodiments, the labelledspecific binding member further includes an acceptor chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith, e.g., the multichromophore is a polymeric tandem dye. Incertain cases, the acceptor chromophore is a fluorophore. In someembodiments of the labelled specific binding member, the acceptorchromophore emission is 1.5-fold greater or more when excited by themultichromophore as compared to direct excitation of the acceptorchromophore with incident light.

In some instances of the labelled specific binding member, themultichromophore is described by formula (VIII):

wherein:

F¹ is a fused 6-5-6 tricyclic co-monomer;

each M¹ and M² are each independently a UV absorbance-modifyingco-monomer;

b is 1 or 2;

a, c, d, e and f are each independently 0 or 1, wherein a+c+d+f≥1;

L¹ is a linking co-monomer that is linked to —Z¹, wherein Z¹ is achemoselective tag or an acceptor chromophore;

n is an integer from 1 to 10,000;

m is an integer from 1 to 10,000;

p is an integer from 1 to 100,000; and

G¹ and G² are each independently selected from a terminal group, a πconjugated segment, a linker and a linked specific binding member,wherein at least one of G¹ and G² is a linked specific binding member.In some instances of formula (VIII), F¹ is a fluorene co-monomer. Incertain instances of formula (VIII), F¹ is a carbazole co-monomer.

In certain embodiments of formula (VIII), the specific binding member isan antibody. In some instances of formula (VIII), the specific bindingmember is an antibody fragment or binding derivative thereof. In somecases of formula (VIII), the specific binding member is an antibodyfragment or binding derivative thereof that is selected from a Fabfragment, a F(ab′)₂ fragment, a scFv, a diabody and a triabody. In someinstances of formula (VIII), the acceptor chromophore is selected from acyanine dye, a xanthene dye, a coumarin dye, a thiazine dye and anacridine dye. In certain instances of formula (VIII), the acceptorchromophore is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640,DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734,DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5 anddiethylamino coumarin.

In some embodiments, the labelled specific binding member is describedby the following structure:

where each R¹ is independently an alkyl substituted with a WSG, or abranching group further substituted with two or more WSGs;

the specific binding member is a biomolecule; and

n is an integer from 1 to 100,000. In certain embodiments, each R¹ groupis a branching group further substituted with two or more WSGs. Incertain instances, the branching group is a substituted aralkyl, such asa substituted benzyl group, which is substituted with two PEG groups.

In certain instances of the fluorene co-monomer of any one of formulae(I)-(VIII), each R¹ or R² sidechain group is a benzyl group substitutedwith one, two or three PEG moieties (e.g., —O(CH₂CH₂O)_(n)R′ where R′ isH or an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16). In certaininstances of the fluorene co-monomer of any one of formulae (II)-(XII),each R¹ or R² sidechain group is a benzyl group substituted with one—O(CH₂CH₂O)_(n)R′ group (e.g., at the 2, 3 or 4 position), where R′ is Hor an alkyl and n is 1-20, e.g., 3-16 such as n is 8-16. In certaininstances of fluorene co-monomer of any one of formulae (II)-(XII), eachR¹ or R² sidechain group is a benzyl group substituted with two—O(CH₂CH₂O)_(n)R′ groups (e.g., at the 2,4-, 3,4- or 3,5-positions),where each R′ is independently H or an alkyl and each n is independently1-20, e.g., 3-16 such as n is 8-16. In certain instances of the fluoreneco-monomer of any one of formulae (II)-(XII), each R¹ or R² sidechaingroup is a benzyl group substituted with three —O(CH₂CH₂O)_(n)R′ groups(e.g., at the 2,4,6-, 2,4,5- or 3,4,5-positions), where each R′ isindependently H or an alkyl and each n is independently 1-20, e.g., 3-16such as n is 8-16. In certain instances of the fluorene co-monomer ofany one of formulae (I)-(VIII), each R¹ or R² sidechain group is a loweralkyl group substituted with a trivalent branching group eachsubstituted with two PEG moieties (e.g., a —CO—NR″₂ or —O(CH₂R″)₂trivalent branching group where each R″ is independently a PEG moiety(e.g., —O(CH₂CH₂O)_(n)R′ where R′ is H or an alkyl and n is 1-20, e.g.,3-16 such as n is 8-16).

It is understood that the polymeric tandem dye of any one of formulae(I) to (VIII) can alternatively be represented by a formula whichindicates what the mol % values for each co-monomer is in the polymer.For example, in some cases, any one of Formulae (I) to (VIII) can berepresented by one of the following formula:G¹-(M¹)_(v)(F¹)_(x)(M²)_(y)(L¹)_(z)-G²G¹-(M¹)_(v)(F¹)_(x)(M²)_(y)-G²G¹-(F¹)_(x)(M²)_(y)(L¹)_(z)-G²G¹-(F¹)_(x)(M²)_(y)-G²

where v, x, y and z are the mol % values of the co-monomers in theconjugated polymer and L¹ can be linked to C¹ or Z¹ (e.g., as describedherein). In some instances of the formulae, v is 1 mol % or more, suchas 5 mol % or more, 10 mol % or more, 15 mol % or more, 20 mol % ormore, or 25 mol % or more. In some instances of the formulae, v is 25mol % or less, such as 20 mol % or less, 15 mol % or less, 10 mol % orless, 8 mol % or less, 6 mol % or less, 5 mol % or less, 2 mol % orless, 1 mol % or less, or even less. In some instances of the formulae,x is 1 mol % or more, such as 2 mol % or more, 3 mol % or more, 4 mol %or more, 5 mol % or more, 10 mol % or more, 15 mol % or more, 20 mol %or more, 25 mol % or more, 30 mol % or more, 35 mol % or more, 40 mol %or more, 45 mol % or more, 50 mol % or more, or even more. In certaininstances of the formulae, x ranges from 1 mol % to 50 mol %, such asfrom 5 mol % to 25 mol % or from 10 mol % to 25 mol %; or such as from 5mol % to 25 mol % or from 10 mol % to 25 mol %; or such as from 1 mol %to 25 mol %, from 1 mol % to 10 mol %, or from 1 mol % to 5 mol %. Insome instances of the formulae, z is 10 mol % or more, such as 15 mol %or more, 20 mol % or more, 25 mol % or more, 30 mol % or more, 35 mol %or more, 40 mol % or more, 45 mol % or more, 50 mol % or more, or evenmore. In some instances of the formulae, z is 25 mol % or less, such as20 mol % or less, 15 mol % or less, 10 mol % or less, 8 mol % or less, 6mol % or less, 5 mol % or less, 2 mol % or less, 1 mol % or less, oreven less. In some instances of the formulae, y is 1 mol % or more, suchas 5 mol % or more, 10 mol % or more, 15 mol % or more, 20 mol % ormore, or 25 mol % or more. In some instances of the formulae, y is 25mol % or less, such as 20 mol % or less, 15 mol % or less, 10 mol % orless, 8 mol % or less, 6 mol % or less, 5 mol % or less, 2 mol % orless, 1 mol % or less, or even less.

It is understood that for any of the structures and formula depictedherein that in some cases of the subject polymeric dyes the end orterminal groups depicted may be located at the opposite ends to thoseshown, e.g., the end groups may be switched. In some embodiments of themultichromophores described herein (e.g., formulae (I)-(VIII), at leastone end group (e.g., L, L², G¹, G², L-Z) is selected from one of thefollowing structures 1-33:

*=site for covalent attachment to unsaturated backbone;wherein R′ is independently H, halogen, C₁-C₁₂ alkyl, (C₁-C₁₂ alkyl)NH₂,C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl, C₁-C₁₂ haloalkyl,C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino, —[CH₂—CH₂]₄—Z¹, or(C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or —COOH; X¹ is —NH₂, —NHCOOH,—NHCOOC(CH₃)₃, —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or—NHCO[CH₂—CH₂—O]_(x′)(CH₂)_(s′)NH₂; r′ is an integer from 1 to 20; andeach s′ is independently an integer from 1 to 20,(CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0 to50, or a benzyl optionally substituted with one or more halogen,hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(y″)CH₃ where each y″ isindependently an integer from 0 to 50 and R′ is different from R;wherein k is 2, 4, 8, 12 or 24; wherein R¹⁵ is selected from groups I-uhaving the structure:

*=site for covalent attachment to backbone.

In some embodiments of the multichromophores described herein (e.g.,formulae (I)-(VIII), at least one end group (e.g., L, L², G¹, G², L-Z)is selected from one of the following structures:

where r is 0 or an integer from 1-50 (e.g., 1-20); k is 0 or an integerfrom 1-50 (e.g., 1-20); R¹ is as defined for any of the fluoreneco-monomers described herein; and R¹⁶ is selected from H, OH, NH₂,—NH(CH₂)r-NH₂, and —NH(CH₂)_(r)COOH.Methods

As summarized above, aspects of the invention include methods ofevaluating a sample for the presence of a target analyte. In someembodiments, the method includes: (a) contacting the sample with apolymeric dye conjugate that specifically binds the target analyte toproduce a labelling composition contacted sample, where the polymericdye conjugate includes: (i) a water soluble light harvestingmultichromophore (e.g., as described herein) including a conjugatedsegment including: a fused 6-5-6 tricyclic co-monomer (e.g., asdescribed herein); and a UV absorbance-modifying co-monomer (e.g., asdescribed herein); where the multichromophore has an ultravioletabsorption maximum; and (ii) a specific binding member (e.g., asdescribed herein); and (b) assaying the labelling composition contactedsample for the presence of a polymeric dye conjugate-target analytebinding complex to evaluate whether the target analyte is present in thesample.

Any convenient method may be used to contact the sample with a polymericdye conjugate that specifically binds to the target analyte to producethe labelling composition contacted sample. As used herein, the terms“polymeric dye conjugate” and “labelled specific binding member” areused interchangeably. In some instances, the sample is contacted withthe polymeric dye conjugate under conditions in which the specificbinding member specifically binds to the target analyte, if present. Forspecific binding of the specific binding member of the conjugate withthe target analyte, an appropriate solution may be used that maintainsthe biological activity of the components of the sample and the specificbinding member. The solution may be a balanced salt solution, e.g.,normal saline, PBS, Hank's balanced salt solution, etc., convenientlysupplemented with fetal calf serum, human platelet lysate or otherfactors, in conjunction with an acceptable buffer at low concentration,such as from 5-25 mM. Convenient buffers include HEPES, phosphatebuffers, lactate buffers, etc. Various media are commercially availableand may be used according to the nature of the target analyte, includingdMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., in some casessupplemented with fetal calf serum or human platelet lysate. The finalcomponents of the solution may be selected depending on the componentsof the sample which are included.

The temperature at which specific binding of the specific binding memberof the conjugate to the target analyte takes place may vary, and in someinstances may range from 5° C. to 50° C., such as from 10° C. to 40° C.,15° C. to 40° C., 20° C. to 40° C., e.g., 20° C., 25° C., 30° C., 35° C.or 37° C. (e.g., as described above). In some instances, the temperatureat which specific binding takes place is selected to be compatible withthe biological activity of the specific binding member and/or the targetanalyte. In certain instances, the temperature is 25° C., 30° C., 35° C.or 37° C. In certain cases, the specific binding member is an antibodyor fragment thereof and the temperature at which specific binding takesplace is room temperature (e.g., 25° C.), 30° C., 35° C. or 37° C. Anyconvenient incubation time for specific binding may be selected to allowfor the formation of a desirable amount of binding complex, and in someinstances, may be 1 minute (min) or more, such as 2 min or more, 10 minor more, 30 min or more, 1 hour or more, 2 hours or more, or even 6hours or more.

Any convenient specific binding members may be utilized in theconjugate. Specific binding members of interest include, but are notlimited to, those agents that specifically bind cell surface proteins ofa variety of cell types, including but not limited to, stem cells, e.g.,pluripotent stem cells, hematopoietic stem cells, T cells, T regulatorcells, dendritic cells, B Cells, e.g., memory B cells, antigen specificB cells, granulocytes, leukemia cells, lymphoma cells, virus cells(e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts,epithelial cells, endothelial cells, and erythroid cells. Target cellsof interest include cells that have a convenient cell surface marker orantigen that may be captured by a convenient specific binding memberconjugate. In some embodiments, the target cell is selected from HIVcontaining cell, a Treg cell, an antigen-specific T-cell populations,tumor cells or hematopoetic progenitor cells (CD34+) from whole blood,bone marrow or cord blood. Any convenient cell surface proteins or cellmarkers may be targeted for specific binding to polymeric dye conjugatesin the subject methods. In some embodiments, the target cell includes acell surface marker selected from a cell receptor and a cell surfaceantigen. In some cases, the target cell may include a cell surfaceantigen such as CD11 b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25,CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34,CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptorgamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5(N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.

Any convenient targets may be selected for evaluation utilizing thesubject methods. Targets of interest include, but are not limited to, anucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, aprotein, such as a fusion protein, a modified protein, such as aphosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylatedprotein, or an antibody, a peptide, an aggregated biomolecule, a cell, asmall molecule, a vitamin and a drug molecule. As used herein, the term“a target protein” refers to all members of the target family, andfragments thereof. The target protein may be any protein of interest,such as a therapeutic or diagnostic target, including but not limitedto: hormones, growth factors, receptors, enzymes, cytokines,osteoinductive factors, colony stimulating factors and immunoglobulins.The term “target protein” is intended to include recombinant andsynthetic molecules, which can be prepared using any convenientrecombinant expression methods or using any convenient syntheticmethods, or purchased commercially. In some embodiments, the polymericdye conjugates include an antibody or antibody fragment. Any convenienttarget analyte that specifically binds an antibody or antibody fragmentof interest may be targeted in the subject methods.

In some embodiments, the target analyte is associated with a cell. Incertain instances, the target analyte is a cell surface marker of thecell. In certain cases, the cell surface marker is selected from a cellreceptor and a cell surface antigen. In some instances, the targetanalyte is an intracellular target, and the method further includeslysing the cell.

In some embodiments, the sample may include a heterogeneous cellpopulation from which target cells are isolated. In some instances, thesample includes peripheral whole blood, peripheral whole blood in whicherythrocytes have been lysed prior to cell isolation, cord blood, bonemarrow, density gradient-purified peripheral blood mononuclear cells orhomogenized tissue. In some cases, the sample includes hematopoeticprogenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cordblood. In certain embodiments, the sample includes tumor cells inperipheral blood. In certain instances, the sample is a sample including(or suspected of including) viral cells (e.g., HIV).

The labelled specific binding members find use in the subject methods,e.g., for labeling a target cell, particle, target or analyte with apolymeric dye or polymeric tandem dye. For example, labelled specificbinding members find use in labeling cells to be processed (e.g.,detected, analyzed, and/or sorted) in a flow cytometer. The labelledspecific binding members may include antibodies that specifically bindto, e.g., cell surface proteins of a variety of cell types (e.g., asdescribed herein). The labelled specific binding members may be used toinvestigate a variety of biological (e.g., cellular) properties orprocesses such as cell cycle, cell proliferation, cell differentiation,DNA repair, T cell signaling, apoptosis, cell surface protein expressionand/or presentation, and so forth. labelled specific binding members maybe used in any application that includes (or may include)antibody-mediated labeling of a cell, particle or analyte.

In some embodiments, the polymeric dye conjugate includes a polymerictandem dye (e.g., as described herein). As such, in some embodiments,the polymeric dye conjugate further includes an acceptor chromophorecovalently linked to the multichromophore in energy-receiving proximitytherewith. In certain embodiments, the conjugate is described by formula(VII):

where:

F¹ is a fused 6-5-6 tricyclic co-monomer;

each M¹ and M² are each independently a UV absorbance-modifyingco-monomer;

b is 1 or 2;

a, c, d, e and f are each independently 0 or 1, wherein a+c+d+f≥1;

L¹ is a linking co-monomer that is linked to —Z¹, wherein Z¹ is achemoselective tag or an acceptor chromophore;

n is an integer from 1 to 10,000;

m is an integer from 1 to 10,000;

p is an integer from 1 to 100,000; and

G¹ and G² are each independently selected from a terminal group, a πconjugated segment, a linker and a linked specific binding member,wherein at least one of G¹ and G² is a linked specific binding member.In some instances of formula (IX), F¹ is a fluorene co-monomer. Incertain instances of formula (IX), F¹ is a carbazole co-monomer.

Once the sample has been contacted with the polymeric dye conjugate, anyconvenient methods may be utilized in assaying the labelling compositioncontacted sample that is produced for the presence of a polymeric dyeconjugate-target analyte binding complex. The polymeric dyeconjugate-target analyte binding complex is the binding complex that isproduced upon specific binding of the specific binding member of theconjugate to the target analyte, if present. Assaying the labellingcomposition contacted sample may include detecting a fluorescent signalfrom the binding complex, if present. In some cases, the assayingincludes a separating step where the target analyte, if present, isseparated from the sample. A variety of methods may be utilized toseparate a target analyte from a sample, e.g., via immobilization on asupport. Assay methods of interest include, but are not limited to, anyconvenient methods and assay formats where pairs of specific bindingmembers such as avidin-biotin or hapten-anti-hapten antibodies find use,are of interest. Methods and assay formats of interest that may beadapted for use with the subject compositions include, but are notlimited to, flow cytometry methods, in-situ hybridization methods,enzyme-linked immunosorbent assays (ELISAs), western blot analysis,magnetic cell separation assays and fluorochrome purificationchromatography.

In certain embodiments, the method further includes contacting thesample with a second specific binding member that specifically binds thetarget analyte. In certain instances, the second specific binding memberis support bound. Any convenient supports may be utilized to immobilizea component of the subject methods (e.g., a second specific bindingmember). In certain instances, the support is a particle, such as amagnetic particle. In some instances, the second specific binding memberand the polymeric dye conjugate produce a sandwich complex that may beisolated and detected, if present, using any convenient methods. In someembodiments, the method further includes flow cytometrically analyzingthe polymeric dye conjugate-target analyte binding complex, i.e., afluorescently labelled target analyte. Assaying for the presence of apolymeric dye conjugate-target analyte binding complex may provide assayresults (e.g., qualitative or quantitative assay data) which can be usedto evaluate whether the target analyte is present in the sample.

Any convenient supports may be utilized in the subject methods. Supportsof interest include, but are not limited to: solid substrates, where thesubstrate can have a variety of configurations, e.g., a sheet, bead, orother structure, such as a plate with wells; beads, polymers, particle,a fibrous mesh, hydrogels, porous matrix, a pin, a microarray surface, achromatography support, and the like. In some instances, the support isselected from a particle, a planar solid substrate, a fibrous mesh, ahydrogel, a porous matrix, a pin, a microarray surface and achromatography support. The support may be incorporated into a systemthat it provides for cell isolation assisted by any convenient methods,such as a manually-operated syringe, a centrifuge or an automated liquidhandling system. In some cases, the support finds use in an automatedliquid handling system for the high throughput isolation of cells, suchas a flow cytometer.

In some embodiments of the method, the separating step includes applyingan external magnetic field to immobilize a magnetic particle. Anyconvenient magnet may be used as a source of the external magnetic field(e.g., magnetic field gradient). In some cases, the external magneticfield is generated by a magnetic source, e.g. by a permanent magnet orelectromagnet. In some cases, immobilizing the magnetic particles meansthe magnetic particles accumulate near the surface closest to themagnetic field gradient source, i.e. the magnet.

The separating may further include one or more optional washing steps toremove unbound material of the sample from the support. Any convenientwashing methods may be used, e.g., washing the immobilized support witha biocompatible buffer which preserves the specific binding interactionof the polymeric dye and the specific binding member. Separation andoptional washing of unbound material of the sample from the supportprovides for an enriched population of target cells where undesiredcells and material may be removed.

In certain embodiments, the method further includes detecting thelabelled target. Detecting the labelled target may include exciting themultichromophore with one or more lasers and subsequently detectingfluorescence emission from the polymeric dye using one or more opticaldetectors.

Also provided are methods of labelled a target molecule. The subjectpolymeric dyes, including tandem dyes, find use in a variety of methodsof labelling, separation, detection and/or analysis. In someembodiments, the method includes: contacting the target molecule with apolymeric dye to produce a labelled target molecule, wherein thepolymeric dye includes: a water soluble light harvestingmultichromophore comprising a conjugated segment comprising: a fluoreneco-monomer; and a UV absorbance-modifying co-monomer; wherein themultichromophore has an ultraviolet absorption maximum; and aconjugation tag that covalently links to the target molecule.

In some embodiments, the polymeric dye is a polymeric tandem dye. Assuch, in some cases, the polymeric dye further includes an acceptorchromophore covalently linked to the multichromophore inenergy-receiving proximity therewith. In certain instances, thepolymeric dye is itself fluorescent. In some embodiments, the polymericdye is described by formula (IX):

where:

F¹ is a fused 6-5-6 tricyclic co-monomer;

each M¹ and M² are each independently a UV absorbance-modifyingco-monomer;

b is 1 or 2;

a, c, d, e and f are each independently 0 or 1, wherein a+c+d+f≥1;

L¹ is a linking co-monomer and Z¹ is a chemoselective tag or an acceptorchromophore;

n is an integer from 1 to 10,000;

m is 0 or an integer from 1 to 10,000;

p is an integer from 1 to 100,000; and

one of G¹ and G² is a terminal group and the other of G¹ and G² is theconjugation tag. In certain embodiments, Z¹ is the acceptor chromophore.In certain embodiments, Z¹ is a chemoselective tag. In certain cases, eis 0. In some instances of formula (X), F¹ is a fluorene co-monomer. Incertain instances of formula (X), F¹ is a carbazole co-monomer.

As used herein the term “conjugation tag” refers to a group thatincludes a chemoselective functional group (e.g., as described herein)that can covalently link with a compatible functional group of a targetmolecule, after optional activation and/or deprotection. Any convenientconjugation tags may be utilized in the subject polymeric dyes in orderto conjugate the dye to a target molecule of interest. In someembodiments, the conjugation tag includes a terminal functional groupselected from an amino, a carboxylic acid or a derivative thereof, athiol, a hydroxyl, a hydrazine, a hydrazide, a azide, an alkyne and aprotein reactive group (e.g. amino-reactive, thiol-reactive,hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive).

Any convenient methods and reagent may be adapted for use in the subjectlabelling methods in order to covalently link the conjugation tag to thetarget molecule. Methods of interest for labelling a target, include butare not limited to, those methods and reagents described by Hermanson,Bioconjugate Techniques, Third edition, Academic Press, 2013. Thecontacting step may be performed in an aqueous solution. In someinstances, the conjugation tag includes an amino functional group andthe target molecule includes an activated ester functional group, suchas a NHS ester or sulfo-NHS ester, or vice versa. In certain instances,the conjugation tag includes a maleimide functional group and the targetmolecule includes a thiol functional group, or vice versa.

Any convenient target molecules may be selected for labelling utilizingthe subject methods. Target molecules of interest include, but are notlimited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA orANA molecule, a protein, such as a fusion protein, a modified protein,such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, oracetylated protein, or an antibody, a peptide, an aggregatedbiomolecule, a cell, a small molecule, a vitamin and a drug molecule. Asused herein, the term “a target protein” refers to all members of thetarget family, and fragments thereof. The target protein may be anyprotein of interest, such as a therapeutic or diagnostic target,including but not limited to: hormones, growth factors, receptors,enzymes, cytokines, osteoinductive factors, colony stimulating factorsand immunoglobulins. The term “target protein” is intended to includerecombinant and synthetic molecules, which can be prepared using anyconvenient recombinant expression methods or using any convenientsynthetic methods, or purchased commercially. In some embodiments, thetarget molecule is a specific binding member (e.g., as describedherein). In certain instances, the specific binding member is anantibody. In some instances, the specific binding member is an antibodyfragment or binding derivative thereof. In some case, the antibodyfragment or binding derivative thereof is selected from a Fab fragment,a F(ab′)₂ fragment, a scFv, a diabody and a triabody.

In some cases, the method includes a separating step where the labelledtarget molecule is separated from the reaction mixture, e.g., excessreagents or unlabeled target. A variety of methods may be utilized toseparate a target from a sample, e.g., via immobilization on a support,precipitation, chromatography, and the like.

In some instances, the method further includes detecting and/oranalyzing the labelled target molecule. In some instances, the methodfurther includes fluorescently detecting the labelled target molecule.Any convenient methods may be utilized to detect and/or analyse thelabelled target molecule in conjunction with the subject methods andcompositions. Methods of analyzing a target of interest that find use inthe subject methods, include but are not limited to, flow cytometry,in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs),western blot analysis, magnetic cell separation assays and fluorochromepurification chromatography. Detection methods of interest include butare not limited to fluorescence spectroscopy, nucleic acid sequencing,fluorescence in-situ hybridization (FISH), protein mass spectroscopy,flow cytometry, and the like.

Detection may be achieved directly via a reporter molecule, orindirectly by a secondary detection system. The latter may be based onany one or a combination of several different principles including butnot limited to, antibody labelled anti-species antibody and other formsof immunological or non-immunological bridging and signal amplificationsystems (e.g., biotin-streptavidin technology, protein-A and protein-Gmediated technology, or nucleic acid probe/anti-nucleic acid probes, andthe like). The label used for direct or indirect detection may be anydetectable reported molecule. Suitable reporter molecules may be thoseknown in the field of immunocytochemistry, molecular biology, light,fluorescence, and electron microscopy, cell immunophenotyping, cellsorting, flow cytometry, cell visualization, detection, enumeration,and/or signal output quantification. Labels of interest include, but arenot limited to fluorophores, luminescent labels, metal complexes,radioisotopes, biotin, streptavidin, enzymes, or other detection labelsand combination of labels such as enzymes and a luminogenic substrate.Enzymes of interest and their substrates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and luciferase, and thelike. More than one antibody of specific and/or non-specific naturemight be labelled and used simultaneously or sequentially to enhancetarget detection, identification, and/or analysis. Labels of interestinclude, but are not limited to FITC (fluorescein isothiocyanate) AMCA(7-amino-4-methylcoumarin-3-acetic acid), Alexa Fluor 488, Alexa Fluor594, Alexa Fluor 350, DyLight350, phycoerythrin, allophycocyanin andstains for detecting nuclei such as Hoechst 33342, LDS751, TO-PRO andDAPI.

Systems

Aspects of the invention further include systems for use in practicingthe subject methods and compositions. A sample analysis system mayinclude a flow channel loaded with a sample and a labelled specificbinding member. In some embodiments, the system is a flow cytometricsystem including: a flow cytometer including a flow path; a compositionin the flow path, wherein the composition includes: a sample; and alabelled specific binding member (e.g., as described herein). In someinstances of the system, the labelled specific binding member includes:a water soluble light harvesting multichromophore including a conjugatedsegment comprising: a fused 6-5-6 tricyclic co-monomer; and a UVabsorbance-modifying co-monomer; where the multichromophore has anultraviolet absorption maximum; and a specific binding member thatspecifically binds a target analyte and is covalently linked to themultichromophore. The multichromophore may be a polymeric dye that isitself fluorescent. The multichromophore may be a polymeric tandem dye.In certain instances, the labelled specific binding member furtherincludes an acceptor chromophore covalently linked to themultichromophore in energy-receiving proximity therewith. In someembodiments, the labelled specific binding member is described byformula (X):

wherein:

F¹ is a fused 6-5-6 tricyclic co-monomer;

each M¹ and M² are each independently a UV absorbance-modifyingco-monomer;

b is 1 or 2;

a, c, d, e and f are each independently 0 or 1, wherein a+c+d+f≥1;

L¹ is a linking co-monomer that is linked to —Z¹, wherein Z¹ is achemoselective tag or an acceptor chromophore;

n is an integer from 1 to 10,000;

m is an integer from 1 to 10,000;

p is an integer from 1 to 100,000; and

one of G¹ and G² is a terminal group and the other of G¹ and G² is alinked specific binding member. In some instances of formula (XI), F¹ isa fluorene co-monomer. In certain instances of formula (XI), F¹ is acarbazole co-monomer.

In certain embodiments of the system, the composition further includes asecond specific binding member that is support bound and specificallybinds the target analyte. In some cases, the support includes a magneticparticle. As such, in certain instances, the system may also include acontrollable external paramagnetic field configured for application toan assay region of the flow channel.

The sample may include a cell. In some instances, the sample is acell-containing biological sample. In some instances, the sampleincludes a labelled specific binding member specifically bound to atarget cell. In certain instances, the target analyte that isspecifically bound by the specific binding member is a cell surfacemarker of the cell. In certain cases, the cell surface marker isselected from a cell receptor and a cell surface antigen.

In certain aspects, the system may also include a light sourceconfigured to direct light to an assay region of the flow channel. Thesystem may include a detector configured to receive a signal from anassay region of the flow channel, wherein the signal is provided by thefluorescent composition. Optionally further, the sample analysis systemmay include one or more additional detectors and/or light sources forthe detection of one or more additional signals.

In certain aspects, the system may further include computer-basedsystems configured to detect the presence of the fluorescent signal. A“computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of the computer-based systems of thepresent invention includes a central processing unit (CPU), input means,output means, and data storage means. A skilled artisan can readilyappreciate that any one of the currently available computer-based systemare suitable for use in the subject systems. The data storage means mayinclude any manufacture including a recording of the present informationas described above, or a memory access means that can access such amanufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g., word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In certain aspects, the system includes a flow cytometer. Flowcytometers of interest include, but are not limited, to those devicesdescribed in U.S. Pat. Nos. 4,704,891; 4,727,029; 4,745,285; 4,867,908;5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791;the disclosures of which are herein incorporated by reference.

Other systems may find use in practicing the subject methods. In certainaspects, the system may be a fluorimeter or microscope loaded with asample having a fluorescent composition of any of the embodimentsdiscussed herein. The fluorimeter or microscope may include a lightsource configured to direct light to the assay region of the flowchannel. The fluorimeter or microscope may also include a detectorconfigured to receive a signal from an assay region of the flow channel,wherein the signal is provided by the fluorescent composition.

Kits

Aspects of the invention further include kits for use in practicing thesubject methods and compositions. The compositions of the invention canbe included as reagents in kits either as starting materials or providedfor use in, for example, the methodologies described above.

A kit may include a water soluble light harvesting multichromophoreincluding a conjugated segment including: a fused 6-5-6 tricyclicco-monomer; and a UV absorbance-modifying co-monomer, wherein themultichromophore has an ultraviolet absorption maximum; and one or morecomponents selected from a polymeric tandem dye, a fluorophore, aspecific binding member, a specific binding member conjugate, a supportbound specific binding member, a cell, a support, a biocompatibleaqueous elution buffer, and instructions for use. In some embodiments ofthe kit, the multichromophore is covalently linked to a specific bindingmember. In some instances, the specific binding member is an antibody.In certain instances, the specific binding member is an antibodyfragment or binding derivative thereof. in certain cases, the antibodyfragment or binding derivative thereof is selected from a Fab fragment,a F(ab′)2 fragment, a scFv, a diabody and a triabody. Themultichromophore may be a polymeric dye that is itself fluorescent. Themultichromophore may be a polymeric tandem dye. In some cases, themultichromophore further includes an acceptor chromophore covalentlylinked to the multichromophore in energy-receiving proximity therewith.

In certain embodiments, the kit finds use in evaluating a sample for thepresence of a target analyte, such as an intracellular target. As such,in some instances, the kit includes one or more components suitable forlysing cells. The one or more additional components of the kit may beprovided in separate containers (e.g., separate tubes, bottles, or wellsin a multi-well strip or plate).

In certain aspects, the kit further includes reagents for performing aflow cytometric assay. Reagents of interest include, but are not limitedto, buffers for reconstitution and dilution, buffers for contacting acell sample the multichromophore, wash buffers, control cells, controlbeads, fluorescent beads for flow cytometer calibration and combinationsthereof. The kit may also include one or more cell fixing reagents suchas paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or anycombinations or buffers thereof. Further, the kit may include a cellpermeabilizing reagent, such as methanol, acetone or a detergent (e.g.,triton, NP-40, saponin, tween 20, digitonin, leucoperm, or anycombinations or buffers thereof. Other protein transport inhibitors,cell fixing reagents and cell permeabilizing reagents familiar to theskilled artisan are within the scope of the subject kits.

The compositions of the kit may be provided in a liquid composition,such as any suitable buffer. Alternatively, the compositions of the kitmay be provided in a dry composition (e.g., may be lyophilized), and thekit may optionally include one or more buffers for reconstituting thedry composition. In certain aspects, the kit may include aliquots of thecompositions provided in separate containers (e.g., separate tubes,bottles, or wells in a multi-well strip or plate).

In addition, one or more components may be combined into a singlecontainer, e.g., a glass or plastic vial, tube or bottle. In certaininstances, the kit may further include a container (e.g., such as a box,a bag, an insulated container, a bottle, tube, etc.) in which all of thecomponents (and their separate containers) are present. The kit mayfurther include packaging that is separate from or attached to the kitcontainer and upon which is printed information about the kit, thecomponents of the and/or instructions for use of the kit.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, DVD, portable flash drive, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the Internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Utility

The compositions, methods and systems as described herein may find usein a variety of applications, including diagnostic and researchapplications, in which the labelling detection and/or analysis of atarget of interest is desirable. Such applications include methodologiessuch as cytometry, microscopy, immunoassays (e.g. competitive ornon-competitive), assessment of a free analyte, assessment of receptorbound ligand, and so forth. The compositions, system and methodsdescribed herein may be useful in analysis of any of a number ofsamples, including but not limited to, biological fluids, cell culturesamples, and tissue samples. In certain aspects, the compositions,system and methods described herein may find use in methods whereanalytes are detected in a sample, if present, using fluorescent labels,such as in fluorescent activated cell sorting or analysis, immunoassays,immunostaining, and the like. In certain instances, the compositions andmethods find use in applications where the evaluation of a sample forthe presence of a target analyte is of interest.

In some cases, the methods and compositions find use in any assay formatwhere the detection and/or analysis of a target from a sample is ofinterest, including but not limited to, flow cytometry, in-situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separation assays and fluorochrome purificationchromatography. In certain instances, the methods and compositions finduse in any application where the fluorescent labelling of a targetmolecule is of interest. The subject compositions may be adapted for usein any convenient applications where pairs of specific binding membersfind use, such as biotin-streptavidin and hapten-anti-hapten antibody.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1

A series of tandem dyes were prepared based on a core Structure 1depicted below including a series of linked fluorophores. FIG. 1illustrates the fluorescence emission profiles of a variety of polymerictandem dyes based on the multichromophore core structure 1 linked to avariety of different acceptor chromophores.

where the Dye is Dyomics dyes DY 431, DY 485XL, DY 610, DY 640, Cy3, Cy3.5 or diethylamino coumarin (DEAC).

A second series of UV absorbing polymeric dyes were prepared andcharacterized. FIG. 2 illustrates the UV absorption spectra of a varietyof multichromophores of interest, MC-1 to MC-5.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the following.

What is claimed is:
 1. A method of evaluating a sample for the presenceof a target analyte, the method comprising: (a) contacting the samplewith a polymeric dye conjugate that specifically binds the targetanalyte to produce a labelling composition contacted sample, wherein thepolymeric dye conjugate comprises: (i) a water-soluble light harvestingmultichromophore comprising a repeat unit comprising: a fused 6-5-6tricyclic co-monomer; and a UV absorbance-modifying co-monomer that is asubstituted aryl co-monomer or a substituted or unsubstituted heteroarylco-monomer; and (ii) a specific binding member covalently linked to themultichromophore; and (b) assaying the labelling composition contactedsample for the presence of a polymeric dye conjugate-target analytebinding complex to evaluate whether the target analyte is present in thesample.
 2. The method according to claim 1, wherein the polymeric dyeconjugate further comprises an acceptor chromophore covalently linked tothe multichromophore in energy-receiving proximity therewith.
 3. Themethod according to claim 2, wherein the acceptor chromophore isselected from cyanine dye, xanthene dye, coumarin dye, thiazine dye andacridine dye.
 4. The method according to claim 1, wherein the fused6-5-6 tricyclic co-monomer comprises a central 5-membered ring that is aheterocycle.
 5. The method according to claim 4, wherein the fused 6-5-6tricyclic co-monomer is a carbazole co-monomer.
 6. The method accordingto claim 1, wherein the fused 6-5-6 tricyclic co-monomer is a fluoreneco-monomer linked via positions 2 and 7 to a polymer backbone.
 7. Themethod according to claim 1, wherein the fused 6-5-6 tricyclicco-monomer is a fluorene co-monomer linked via positions 3 and 6 to apolymer backbone.
 8. The method according to claim 6, wherein the fused6-5-6 tricyclic co-monomer is of the structure:

wherein: Z is C(R¹)₂ or —N(R¹)—; each R is H; and each R¹ isindependently selected from alkyl, substituted alkyl, aralkyl,substituted aralkyl, PEG moiety, water solubilizing group and -L¹-Z¹,where L¹ is a linker and Z¹ is a chemoselective tag.
 9. The methodaccording to claim 8, wherein each R¹ is substituted alkyl comprisingone or more water solubilizing groups or substituted aralkyl comprisingone or more water solubilizing groups.
 10. The method according to claim1, wherein the UV absorbance-modifying co-monomer is selected fromsubstituted 1,4-phenyl, substituted 1,3-phenyl, substituted orunsubstituted 2,5-pyridyl, and substituted or unsubstituted 2,6-pyridyl.11. The method according to claim 1, wherein the UV absorbance-modifyingco-monomer is selected from one of the following:

wherein: Z²-Z⁵ are each independently CR or N, wherein at least oneZ²-Z⁵ is N; and each R and R¹¹-R¹⁶ are independently selected from thegroup consisting of hydrogen, halogen, cyano, alkoxy, substitutedalkoxy, alkyl and substituted alkyl.
 12. The method according to claim11, wherein the UV absorbance-modifying co-monomer is selected from oneof the following:

wherein n is 1-20 and R′ is H or lower alkyl.
 13. The method accordingto claim 11, wherein the substituted aryl co-monomer is of thestructure:

wherein R¹¹ and R¹³ are each independently an alkyl or a substitutedalkyl.
 14. The method according to claim 11, wherein the substitutedaryl co-monomer is of the structure:

wherein R¹¹ is an alkyl or a substituted alkyl.
 15. The method accordingto claim 1, wherein: the multichromophore comprises 25% or more bymolarity of the UV absorbance-modifying co-monomer; and themultichromophore is a conjugated polymer comprising 5 or more monomericrepeat units.
 16. The method according to claim 1, wherein themultichromophore has an absorption maximum wavelength in the range of300 to 400 nm and an emission maximum wavelength in the range of 375 to900 nm, wherein 80% or more of the integrated absorption intensity is inthe UV region.
 17. The method according to claim 1, wherein the targetanalyte is associated with a cell.
 18. The method according to claim 17,wherein the target analyte is a cell surface marker of the cell.
 19. Themethod according to claim 17, wherein the target analyte is anintracellular target, and the method further comprises lysing the cell.20. The method according to claim 1, wherein the method furthercomprises flow cytometrically analyzing the polymeric dyeconjugate-target analyte binding complex.